Using Proteomics to Elucidate Critical Signaling Pathways Involved in Hematopoietic Differentiation and Migration

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Using Proteomics To Elucidate Critical Signaling Pathways Involved In Hematopoietic Differentiation And Migration

Thesis by

Heba Ahmed

In partial fulfillment of the requirements

For the degree of

Master of science

King Abdullah University of Science and Technology

Thuwal, Kingdom of Saudi Arabia

November 2012

EXAMINATION COMMITTEE APPROVALS FORM

The thesis of Heba Ahmed is approved by the examination committee.

Committee Chairperson: Jasmeen Merzeban

Committee Member: Christopher Gehring

Committee Member: Samah Zeineb Gadhoum

Heba Ahmed

ABSTRACT

Using Proteomics To Elucidate Critical Signaling Pathways Involved In Hematopoietic Differentiation And Migration

Heba Ahmed

Despite important advances in the therapy of acute myeloid leukemia

(AML) the majority of patients will die from their disease (Appelbaum,

Rowe, Radich, & Dick, 2001). Characterization of the aberrant molecular pathways responsible for this malignancy provides a platform to discover alternative treatments to help alter the fate of patients.

AML is characterized by a blockage in the differentiation of myeloid cells resulting in the accumulation of highly proliferating immature hematopoietic cells. Since treatments such as chemotherapy rarely destroy the leukemic cells entirely, differentiation induction therapy has become a very attractive treatment option. Interestingly, previous experiments have shown that ligation of CD44, a cell surface glycoprotein strongly expressed on all AML cells, with anti-CD44 monoclonal antibodies (mAbs) could reverse this block in differentiation of leukemic blasts regardless of the

AML subtype. To expand the understanding of the cellular regulation and 5 circuitry involved, we aim to apply quantitative phosphoproteomics to monitor dynamic changes in phosphorylation state in response to anti-

CD44 treatment.

Protein phosphorylation and dephosphorylation is a highly controlled biochemical process that responds to various intracellular and extracellular stimuli. As phosphorylation is a dynamic process, quantification of these phosphorylation events would be vastly insightful.

The main objective of this project is to determine the differentiation- dependent phosphoproteome of AML cells upon treatment of cells with the anti-CD44 mAb.

In these experiments, optimization of protein extraction, phosphopeptide enrichment and data processing and analysis has been achieved. The primary results show successful phosphoproteome extraction complemented with efficient phosphopeptide enrichment and informative data processing. Further quantification with stable isotope labeling techniques is anticipated to provide candidates for targeted therapy. 6

ACKNOWLEDGEMENTS

Foremost, I would like to express my sincere gratitude to my Supervisor Professor:

Jasmeen Merzaban for the continuous support of my study and research, for her patience, motivation, enthusiasm, and immense knowledge. And thanks to Dr. Zeineb

Gadhoum and Dr. Kosuke Sakashita for their help and guidance throughout the time of my research.

I would also like to thank my committee member Professor Chrisopher Gehring for his guidance and support during my Masters education.

I thank my fellow lab mates, Dina Abu Samra, Nour Madhoun, Amal Ali and Ayman El

Khodiery for bearing with all my endless questions interrupting their lab work, for our stimulating discussions, and for all the fun we have had.

Thanks to Maryame Mih for her great efforts in managing the lab and providing the atmosphere for productive work.

Last but not least, I owe my loving thanks to my husband and lovely daughters who endured so much for me. And special gratitude goes to my Father and rest of my family members for their constant support and encouragement throughout my research work.

And above all I thank Allah

Finally, I thank all those who have helped me directly or indirectly in the successful completion of my thesis. Anyone missed in this acknowledgement are also thanked. 7

TABLE OF CONTENTS

EXAMINATION COMMITTEE APPROVALS FORM ...... 2 ABSTRACT ...... 4 ACKNOWLEDGEMENTS ...... 6 TABLE OF CONTENTS...... 7 LIST OF ABBREVIATIONS ...... 8 LIST OF ILLUSTRATIONS...... 10 LIST OF TABLES ...... 11 1. INTRODUCTION ...... 12 1.1 Leukemia: Blockage in Hematopoietic differentiation ...... 12 1.2 Differentiation therapy ...... 16 1.3 Phosphoproteomics and SILAC Labeling ...... 18 2. MATERIALS AND METHODS ...... 24 2.1 Cell Culturing and lysis ...... 24 2.2 Gel preparation and staining ...... 26 2.3 Protein digestion and desalting...... 27 2.4 Off-gel fractionation, Strong cation exchange chromatography and Phosphopeptide enrichment ...... 28 Off-gel fractionation ...... 28 2.5 Mass spectrometric analysis and data processing ...... 30 3. RESULTS...... 33 3.1 Quality of Lysate ...... 33 3.2 Protein Coverage of different lysate Portions: ...... 35 3.3 Enrichment Efficiency ...... 38 3.4 Phosphorylated proteins found in Kg1a cells ...... 38 3.5 Phosphorylated Proteins found in HL60 cells ...... 43 4. DISCUSSION ...... 45 BIBLIOGRAPHY ...... 49 APPENDICES ...... 52

LIST OF ABBREVIATIONS

AML Acute Myeloid Leukemia

AS2O3 Arsenic Trioxide

BM Bone Marrow

CAN Acetonitril

CD34, CD38, CD44 Cluster of Differentiation

Cys Cysteine

Da Dalton

DTT Dithiothreitol

EGF Epidermal Growth Factor

FBS Fetal Bovine Serum

G-CSF Granulocyte Colony Stimulating Factor

GO Gene ontology

HAcO Acetic acid

HPLC High Performance Liquid Chromatography

HSC Hematopoietic stem cell

IFNa Interferon alpha

IMAC Immobilized Metal Affinity Chromatography

IOA Iodoacetamide

LC Liquid Chromatography

LC-MS/MS Liquid Chromatography coupled with Mass Spectroscopy 9

LSC Leukemic Stem Cells mAb monoclonal Antibody

MAPKKK Kinase in the Mitogen-Activated Protein Kinase pathway

Met Methionine

MS Mass spectroscopy pI Iso-electric point

PTM Posttranslational modification pTyr phosphorylated Tyrosine

SDS-PAGE Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis

Ser Serine

SILAC Stable isotope labeling in Amino acid Culture

TEAB Triethyl Ammonium Bicarbonate buffer

TFA Trifluoroacetic acid

Thr Threonine

TiO2 Titanium dioxide

Tyr Tyrosine

LIST OF ILLUSTRATIONS

Figure 1.1: Conventional view of hematopoiesis .) ...... 14 Figure 1.2 Diagram of normal myeloid development and the relationship to leukemic cells and LSCs.

Figure 1.3 Experimental outline for SILAC-based phophoproteomics.

Figure 3.1 Gel Staining Proteins are loaded onto a SDS-page gels (4- 10 %) and subjected to SDS- PAGE electrophoresis.

Figure 3.2: Flow diagram of the Phosphoproteomics platform applied ...... 37

Figure 3.3 Proportion of singly and multiply phosphorylated peptides in Kg1a cells...... 39

Figure 3.4 Distribution of identified phosphorylated serine, threonine, and tyrosine residues in resting Kg1a cells...... 39

Figure 3.5 Frequency of phosphorylated proteins identified with selected GO biological process in Kg1a cells...... 40

Figure 3.6 Frequency of phosphoproteins identified in HL60 cell-line involved in differentiation and hemopoiesis...... 44

LIST OF TABLES

Table 3.1: A list of the proteins present in the detergent-insoluble portion of the lysate...... 37

1. Introduction

1.1 Leukemia: Blockage in Hematopoietic differentiation

Leukemia has been renowned as the type of cancer that can be cured with a single transplantation of bone marrow (BM). It depends on the notion that irradiation of diseased cells and replacing them with healthy counterparts should provide sufficient clinical therapy. Indeed, the standard treatment protocol for treatment of AML has been chemo- radiotherapy prior to BM transplantation which should provide healthy blood cells. Unfortunately, following this strategy, only about one-third of those between ages 18–60 who are diagnosed with AML can be cured; disease-free survival is rare and current therapy devastating in older adults

(Stone, Donnell, & Sekeres, n.d.). Advances in the understanding of the pathophysiology and clinical aspects of acute myeloid leukemia (AML) have not led to major improvements in disease-free and overall survival of patients with this disease obligating a more cellular and molecular definition and classification.

Different theories have been proposed for defining the major contributors of tumorigenesis. From the possibility that cell-cycle loss of 13 control can solely cause disruption of differentiation mechanisms

(Chow, A. Y. (2010) or if they are affected by secondary factors leading to cancer formation to the notion that both cell cycle and differentiation are affected by other gene products either dependently or independently. All these hypothesis share a common fact that differentiation blockage is the major contributor.(Tenen, 2003)

Hematopoietic differentiation fidelity dictates the abundance and functionality of the different cell types in blood. Hematopoiesis is characterized by finely coordinated process of continuous blood cell formation starting at a common multipotent hematopoietic stem cell (HSC), which is defined by both its capacity of self-renewal and the capacity to generate an exact replicate of itself (a sister cell) that will undergo differentiation. This later will give rise to precursor cells that will lose this capacity of self-renewal but acquire a strong capacity of proliferation. In normal hematopoiesis, this differentiation process will lead to lineage- committed and fully functional cells generation that ultimately loses all proliferative capacity as it reaches complete maturation.(Metcalf, 2007) 14

Figure 1.1: Conventional view of hematopoiesis . Multipotential stem cells are self-renewing cells that have the ability to produce precursor cells that are more lineage restricted and have an increased proliferative potential. Within each lineage, population cell numbers rise with increasing maturity. Adapted from (Metcalf, 2007) When this tightly controlled phenomenon is disrupted and the differentiation process is arrested, it leads to the accumulation in the blood stream of nonfunctional immature blood cells that did not lose their proliferation capacity. These cells will progressively invade the blood stream resulting in the heterogeneous disease defined as AML. The French

American British classification separated AML into different subclasses according to the stage of differentiation blockage and type of blast accumulations. (Figure 1.2) 15

Figure 1.1 Diagram of normal myeloid development and the relationship to leukemic cells and LSCs. The malignant cells in acute and chronic myeloid leukemia are indicated in red boxes and classified by FAB classification according to the stage of blockage of differentiation. The LSCs are restricted to rare multipotent committed progenitors indicated with the blue boxes. Adapted from (Krause & Van Etten, 2007) The conventional treatment of AML remains chemotherapy that targets highly proliferating cells (blasts). The high incidence of relapse after this conventional treatment has led to the hypothesis of the presence of a subpopulation of cells that reside in the bone marrow. These cells which are resistant to chemotherapy drugs (because of the quiescence state) are capable of repopulating into leukemic blasts dominating the bone marrow niche after treatment. This rare population of cells called leukemic stem 16 cells (LSCs) have a phenotype that is very similar to the normal stem cells

(HSCs) and, just as the HSCs, were characterized by their infinite ability of self-renewal and surface phenotype CD34+CD38-(Reya, Morrison, Clarke, &

Weissman, 2001). Because the dormancy or the long-term quiescence of these cells is thought to be the main reason behind their resistance, recent studies aim to target them by breaking their dormancy using different differentiating agents such as Interferon-alpha(IFNa) and G-CSF as well as arsenic trioxide (As2O3) followed by targeted chemotherapy in a sequential two-step protocol.(Essers & Trumpp, 2010) The main problem with these agents is that they target both the leukemic and normal stem cell populations.(Jordan, 2008) Consequently, new strategies that specifically and preferentially target the malignant stem cell population, while sparing normal stem cells are required.

1.2 Differentiation therapy

Without a doubt, reversing the blockage in differentiation of leukemic cells is a main point of interest in cancer research, especially after the successful use of Retinoic acid for the treatment of acute promyelocytic leukemia (AML3).(Shu-rong, Jia-xiang, & Long-jun, 2012) In search for other 17 potential targets for differentiation induction, CD44 surfaced as a candidate. CD44 is a cell surface antigen that is expressed on all types of hematopietic cells. However, its expression has been found to be higher on

AML cells compared to their normal counterpart. Importantly, CD44 has been involved in multiple cellular functions such as cell proliferation, cell differentiation, immune response and hematopoiesis.

The effect of CD44 ligation (with anti-CD44 mAbs or with its natural ligand, hyaluronic acid) was experimented both in vitro and in vivo and proven efficient in initiating differentiation of all AML subtypes.(Charrad et al., 1999) Importantly, in addition to being effective in inducing differentiation of all AML subtypes, it is a selective treatment that specifically targets the cancer cells leaving the normal “healthy” cells unaffected. Another very lucrative characteristic of this treatment is its ability to target the rare, slowly dividing Leukemic stem cells (LSCs) in addition to the leukemic blasts. This is imperative since it is well supported that this rare population of LSCs is responsible for the relapse following chemotherapy treatment for AML. By understanding the downstream 18 effectors upon CD44 ligation, we can better devise treatment régimes that specifically target these leukemic blasts and LSCs.

These mechanisms are highly complex and involve interconnected molecular effectors demanding robust techniques to give valuable accurate information that can be used for reaching a successful treatment.

Comparative analysis of the molecular pathways before and after CD44 ligation and mapping the signaling perturbations unique in AML is the main goal of this work. Analyzing the molecular pathways activated/repressed in

LSCs before and after treatment with anti-CD44 and comparing them to the level of activation of the molecular pathways in normal HSCs could provide the key for cancer therapy specificity.

1.3 Phosphoproteomics and SILAC Labeling

Protein phosphorylation is a key posttranslational modification, which reversibly regulates almost all processes in the living cell and as deregulated signaling is a hallmark of cancer and other diseases, studying 19 such an influential process is anticipated to reveal important hidden information about diseases and their etiologies. Improvements in fractionation, enrichment, instrumentation, and data processing have facilitated the application of mass spectrometry–based approaches for examining phosphorylation (Macek, Mann, & Olsen, 2009) Combining phosphoproteomics with stable isotope labeling by amino acids in cell culture (SILAC)(Ong, 2002) has been reported as an effective strategy for quantifying phosphorylation changes on a global scale. It would provide a source of information on the complex and dynamic processes underlying differentiation. Here we aim to characterize the signaling transduction modifications induced in AML before and after CD44 ligation using anti-

CD44 mAbs. Regulatory protein phosphorylation is a transient modification that is often of low occupancy or “stoichiometry,” meaning that only a fraction of a particular protein may be phosphorylated on a given site at any particular time, and that occurs on regulatory proteins of low abundance, such as protein kinases and transcription factors. (Vermeulen et al., 2010) As a result of this phenomenon, efficient phosphopeptide- enrichment methods are imperative in order to determine these changes with sensitivity and accuracy from a complex mixture of proteins present in 20 whole cell lysates. In the initial stage of this study we aimed to determine the phosphoproteome of untreated leukemic cell lines (KG1a and HL60).

The methodology involved in the peptide fractionation (by off-gel isoelectric point fractionation) and phosphopeptide enrichment (by titanium dioxide (TiO2)) will subsequently be used to do further quantitative analysis (i.e. SILAC) of these cells before and after treatment with anti-CD44 antibodies (Figure 3.2). The enriched phosphopeptide fractions were separated by online nanoscale reversed-phase high-pressure liquid chromatography (HPLC) directly coupled to a high-performance mass spectrometer. Phosphopeptides were ionized and analyzed by high- resolution MS using orbitrap mass analyzers. This work scheme has previously been used in combination with (SILAC, Stable Isotope Labeling by amino acids in cell culture. and generated a global view of dynamic regulation of phosphorylation in mammalian cells as a function of a growth factor [epidermal growth factor (EGF)] stimulus in a time-resolved manner

(Olsen et al., 2006).

In the work discussed here, we optimized protein extraction and phosphopeptide enrichment methods, along with bioinformatics analysis pipeline prior to direct application on quantifiable and different time-points of treated samples. Stable Isotope Labeling by Amino acids in Cell culture

(SILAC)(Ong, 2002) will be later used to differentially label control and differentiated cells. This technique utilizes MS to detect quantitative differences in protein levels between samples (Figure 1.3). Two cell populations would be grown in identical medium containing either stable light or heavy isotopes amino acids. After a certain number of doublings, the isotope-labeled amino acids are metabolically incorporated into every protein of the cell. Upon differentiation treatment in the heavy-labeled cell population, equal amounts of cells from the two populations are combined, followed by standard procedures for sample preparation for MS analysis.

The metabolic incorporation of isotope-labeled amino acids leads to a mass shift in the corresponding peptides, with the ratio of the peak intensities reflecting the relative protein amount. This differential peak intensity provides a wealth of information indicating changes in protein expression associated with anti-CD44 mAbs-induced processes and modulation of phosphorylation levels in the underlying signaling pathways. To validate 22 that the differential protein expressions are due to the anti-CD44 mAbs treatment and not the use of heavy isotopes, reciprocal labeling experiment will be carried out where the light-labeled cells are treated while the heavy-labeled cells serve as control.

Figure 1.3 Experimental outline for SILAC-based phophoproteomics. (1) AML cell lines (HL60) are cultured in either light or heavy amino acid for 6 passages to allow full incorporation of the isotopes. (2) Anti-CD44 (or HA) treatment is applied to the heavy-labeled cells and harvested at various time points. (3) The treated heavy labeled cells are mixed 1:1 in cell number to the control, light-labeled cells. (4) The mixed lysate are process with SDS-PAGE and the resulting gel lane is equally sliced into 15-20 pieces to extract protein from them. (5) Prior to MS analysis, the extracted proteins need to go through multiple phosphopeptide enrichment steps. This step is especially crucial for achieving comprehensive and unambiguous detection of phosphopeptides. (6) The ratio of peak intensities coming from the light and heavy-labeled peptides is used to deduce differential protein expression and post-translational modifications.

2. MATERIALS AND METHODS

2.1 Cell Culturing and lysis

KG1a, a human AML-derived HSPC-like CD34+ cell line and HL60, a leukemic cell line used as a model system for studying AML, were purchased (ATCC, Manassas, VA) and cultured in complete RPMI 1640 medium (Invitrogen) supplemented with FBS, Fetal Bovine Serum USA origin (Invitrogen) and 1% Pencillin-streptomycin antibiotic solution (Hyclone, Alliance global).

Cell Lysis

300*106 KG1a Cells were harvested in 3 ml NP40 Lysis buffer

(Invitrogen) containing 1% NP-40, 250 mM NaCl, 5 mM EDTA, 50 mM tris

(pH 7.4), 1 mM sodium orthovanadate, 50 mM sodium fluorate, phosphatase inhibitors (Fisher) and phenylmethanesulfonyl fluoride (Sigma

Aldorich) and protease inhibitor cocktail tablet (Roche Molecular

Biochemicals, Indianapolis, IN). Cells were washed three times with ice- chilled phosphate buffer saline (10 mM phosphate, 150 mM sodium chloride, pH 7.2 from Invitrogen) and centrifuged at 1400rpm at 4oC for 5 minutes using micro centrifuge (Eppendorf) between each wash. They were lysed at 40C for 1 hour with continuous rotation. Protein extracts were clarified by centrifugation at 12,000 rpm to pellet chromatin and other insoluble material. The insoluble pellet was re-dissolved in 8 M urea, 2M 25 thiourea and 1% SDS supplemented with phosphatase inhibitors and protease inhibitors. The soluble proteins in the extract were precipitated overnight at −20°C by addition of four volumes of ice-cold acetone.

Following centrifugation, precipitated proteins were re-dissolved in 8 M urea, 2M thiourea and 1% SDS supplemented with phosphatase inhibitors and protease inhibitors. The protein concentrations of all the fractions were determined with the Bradford assay using plate-reader and evaluated by

SDS–polyacrylamide gel electrophoresis (SDS-PAGE) stained with SYPRO-

Ruby protein gel stain (Invitrogen) for total proteins and Pro-Q diamond phosphoprotein gel stain (Invitrogen) for phosphoproteins.

HL60 cells were Lysed using SILAC protein identification and quantification kit Protocol ( invitrogen , user manual (Manual part no. 25-

0841) preparing cell lysate section). 200x106 HL60 cells were washed three times with ice-chilled phosphate buffer saline (10 mM phosphate, 150 mM sodium chloride, pH 7.2 from Invitrogen) and centrifuged at 1400rpm at 4oC for 5 minutes using micro centrifuge (Eppendorf) between each wash. Cells were then harvested using 1 ml of SILAC kit Lysis buffer A. The pellet was sonicated for 3 X 10 second intervals followed each time by a 1 minute 26 incubation on ice. The sample was further centrifuged at 10,000g for 5 minutes to remove any particles. The supernatant was further collected and protein concentration was measured using Bradford assay using plate- reader. 4 mg protein was used for the rest of the experimental steps.

Acetone precipitation was performed using 4 volumes of acetone at -20oC overnight. Protein collection from acetone precipitation involved centrifugation at 4000 rpm, discarding the supernatant and letting the pellet dry. The protein pellet was then re-dissolved in 500 ul 8M Urea buffer supplemented with phosphatase and protease inhibitors using sonication for 10 seconds followed by 1 minute in ice repeated 3 times.

2.2 Gel preparation and staining

Proteins suspended in Urea/Thiourea buffer where reduced by the addition of 4X Laemmli buffer;NuPAGE® LDS Sample Buffer (4X) (invitrogen) and 10% 2-mercaptoethanol (Fisher chemical)followed by 10 minutes heating at 70°C then loaded onto sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), 4-20% mini-protean TGX (Bio-Rad Laboratories, Hercules,

CA). Electrophoresis was performed for 1h at 120V. Proteins were then fixed in gel by keeping it in 50% methanol/10% acetic acid mixture for 1 27 hour then stained with Pro-Q diamond phosphoproteins gel stain

(Invitrogen), de-stained, washed twice and then imaged using Typhoon scanner (Variable mode imager, GE Healthcare). The same gel is then stained with SYPRO ruby protein gel stain (Invitrogen), de-stained, washed twice and imaged again with Typhoon scanner. (Agrawal & Thelen, 2005)

2.3 Protein digestion and desalting

The proteins dissolved in urea lysis buffer, were reduced by adding 5 mM DTT, Dithiothreitol (Fisher) and incubated for 2 hrs at 30C. The proteins were further alkylated by adding 14 mM IOA, Iodoacetamide and incubated for 30 min in the dark at room temperature followed by quenching un-reacted IOA by increasing DTT concentration to 10 mM, incubated for 15 min in the dark at room temperature, then protein samples were diluted with 50 mM Triethyl Ammonium Bicarbonate (TEAB) buffer (pH 8.5) and finally digested by adding 200 ug trypsin (two vials; sequencing grade modified trypsin, Promega, Madison, WI) prior to incubating the mixture overnight at 30C with gentle agitation. The 28 following day in order to stop the reaction, Trifluoroacetic acid (TFA) is added until a pH<2.0 is reached.

Desalting the solutions using Sep-Pak Vac C18 columns (Waters) was then performed. 100% Acetonitril, 0.5% Acetic acid (HAcO) in 50% ACN then 0.1% TFA were used for conditioning of the column. Samples were treated with TFA to 0.1% then loaded on the column, after which the column was washed with 0.1% TFA followed by 0.5% HAcO. Finally, the peptides were eluted using 50% ACN and 0.5% HAcO mixture. The eluted peptides were then dried using a speed Vacuum (Concentrator plus from eppindorf).

2.4 Off-gel fractionation, Strong cation exchange chromatography and Phosphopeptide enrichment

Off-gel fractionation

KG1a Peptide Samples (proteins after digestion) from both detergent- soluble and insoluble portions of the lysate were subjected to fractionation using agilent 3100 OFFGEL fractionator. It is a technique that separates proteins or peptides according to their isoelectric points. Procedures were 29 conducted following user manual guide (© Agilent Technologies, Inc. 2010 .

12 fractions were collected for each sample. (Detergent-soluble and insoluble samples)

Strong Cation Exchange chromatography

HL60 Peptide samples (Proteins after digestion) were subjected to SCX

(strong cation exchange chromatography).

Workflow. A total of 4 mg of peptide was fractionated using a

PolySULFOETHYL A column (4.6 mm × 100 mm, 5 μm particle size, 200 Å pore size) (PolyLC, Columbia, MD) on a High Speed LC system (Thermo

Scientific, Accela). A 60 min shallow gradient designated for phosphopeptide separation using a combination of 5 mM KH2PO4 in 30% acetonitrile, pH 2.65 (Buffer A) and Buffer A with 350 mM KCl, pH 2.65

(Buffer B) was created.The gradient was composed of 100% A for 5 min; then 0-21% B for 35 min; followed by 21-100% B for 5 min; then maintained at 100% B for 10 min; finally ending at 100% A for 5 min. The column was conditioned in 100% A for 30 min after each gradient and before the next run to ensure reproducibility. The UV detection was monitored at a wavelength of 214 nm. A total of 20 fractions (out of 60 fractions) were 30 collected by pooling most fractions at 2 min intervals and a few (beginning and ending fractions) at 3 min intervals. The fractions were further dried via speed Vacuum, and desalted using SEP-PAK C18 cartridges (Waters

Corporation, Milford, MA) prior to phosphopeptide enrichment.(Gan, Guo,

Zhang, Lim, & Sze, 2008)

Phospopeptide Enrichment

All 24 fractions of peptide samples resulting from off-gel fractionation were subjected to phosphopeptide enrichment system using TiO2 spin tips sample kit (Protea). Standard supplied protocol steps were conducted. The eluted peptides were finally dried using a speed vacuum.

2.5 Mass spectrometric analysis and data processing

Mass Spectrometric Analysis

LC-MS/MS data were collected using LTQ-Orbitrap Velos mass spectrometer (Thermo) coupled to Proxeon nano-LC system. Peptides were captured in a C18 trap column followed by elution via an integrated nanobore column (100 μm × 150 mm, C18 - 3 μm particles, 200 Å pore size,

Michrom BioResources). The peptides were eluted using linear gradients of

5-40% Acetonitrile (in 0.1% formic acid) for 40 min, followed by 40-90% 31

Acetonitrile (0.1% formic acid) for 5 min, and then isocratic gradient of 90% acetonitrile (0.1% formic acid) for 20 min.

The MS was operated in the data dependent mode using captive spray source, electrospray voltage potential of 1.5 kV, ion transfer tube temperature of 160°C. A full survey MS scan (350−1600 m/z range) was acquired in the Orbitrap at a resolution of 100000 and a maximum ion accumulation time of 1000 ms. Precursor ion charge state screening was activated. The linear ion trap was used to collect peptides where 10 most intense ions were selected for collision-induced dissociation (CID) in MS2, which were performed concurrently with a maximum ion accumulation time of 200 ms. A MS3 scan was followed after each MS2 scan when a neutral loss at 97.97 Da was detected. Dynamic exclusion was activated for this process, with a repeat count of 1 and exclusion duration of 30 s.

Data Analysis

The MS spectra were extracted from RAW data files using Proteome

Discoverer 1.2 and converted into MASCOT generic file format, and searched against Human Uniprot database using in-house MASCOT server.

The search was limited to maximum 1 missed trypsin cleavages; #13C of 1; 32 mass tolerances of 10 ppm for peptide precursors; and 0.5 Da mass tolerance for fragment ions. Fixed modification was carbamidomethyl at

Cys residue, whereas variable modifications were oxidation at Met residue, and phosphorylation at Ser, Thr or Tyr residues. Mascot output file was processed and filtered using Scaffold program, and proteins with minimum probability of 99% and minimum peptide probability of 95% were considered positive. Further data filtration was performed using Scaffold

PTM program for identification of phosphopeptides, and assignment of phosphorylation sites.

3. Results

All results shown are for untreated leukemic cell lines for optimization of methods proposed to isolate phosphopeptides within our samples. KG1a cells are a leukemia patient-derived, HSPC-like CD34+ cell line, which serves as a great in vitro model system to characterize the primitive leukemic/hematopoietic cells. Preliminary results for HL60, a leukemic cell line used as a model system for studying AML, are also shown.

3.1 Quality of Lysate

The first step of any proteomics approach is to ensure the best extract and preservation of the proteins isolated from your cell samples. A number of optimization steps are required in order to reach the appropriate cell count: lysis buffer ratio suitable for the cell line in question focusing on determining a manageable protein concentration and amount whilst ensuring total proteome extraction and Post translation modification

(PTM) preservation. As shown in Figure 3.2, the cells were lysed using a 34 non-ionic detergent (NP40) and the extracted proteins were subsequently dissolved in urea/thiourea buffer.

The quality of the lysate was checked with Bradford protein concentration measurement protocols coupled with Gel staining of the urea/thiourea based proteins using SYPRO for total protein staining and

Pro-Q stain for specific staining of phosphoproteins. Figure 3.1 shows efficient protein extraction as the staining gives good band intensities.

Proteome and phosphoproteome coverage is indicated by the large number of bands covering a wide range of molecular weights.

SYPRO staining Pro-Q diamond staining

Figure 3.1 Gel Staining Proteins are loaded onto a SDS-page gels (4- 10 %) and subjected to SDS-PAGE electrophoresis. Proteins are fixed in the gel using a 50% methanol/10% 35 acetic acid mixture and subsequently the gels are stained overnight first with either (A) SYPRO Ruby protein gel stain or (B) Pro-Q diamond phosphoprotein gel stain. After de- staining with 10% methanol 7% acetic acid followed by washing the gels with water, they were scanned with a Typhoon imager.

3.2 Protein Coverage of different lysate Portions:

Insoluble versus detergent-soluble portions

The implemented work scheme generated two separate portions with different protein content. As shown in Figure 3.2, the first step of Lysis separates the proteins according to their solubility in the lysis buffer used.

Running them separately on the orbitrap MS/MS system provided valuable information. A total of 1391 peptides corresponding to 347 proteins were identified in the soluble portion against 29 peptides corresponding to 11 proteins in the insoluble portion.

Protein name Accession number Number of Sequence unique peptides Coverage

Nucleolin OS=Homo sp|P19338|NUCL_HUMAN 5 6.48% sapiens GN=NCL PE=1 SV=3

60 kDa heat shock protein, sp|P10809|CH60_HUMAN 2 3.84% mitochondrial OS=Homo sapiens GN=HSPD1 PE=1 SV=2

Heterogeneous nuclear sp|P09651|ROA1_HUMAN 1 4.84% ribonucleoprotein A1 OS=Homo sapiens GN=HNRNPA1 PE=1 SV=5

Isoform Short of sp|Q00839- 1 2.23% Heterogeneous nuclear 2|HNRPU_HUMAN,sp|Q008 ribonucleoprotein U 39|HNRPU_HUMAN OS=Homo sapiens GN=HNRNPU

Isoform 2 of sp|P52272- 2 3.18% Heterogeneous nuclear 2|HNRPM_HUMAN,sp|P522 ribonucleoprotein M 72|HNRPM_HUMAN OS=Homo sapiens GN=HNRNPM

Heterogeneous nuclear sp|P22626|ROA2_HUMAN 3 14.20% ribonucleoproteins A2/B1 OS=Homo sapiens GN=HNRNPA2B1 PE=1 SV=2

L-lactate dehydrogenase A sp|P00338|LDHA_HUMAN,tr 1 2.71% chain OS=Homo sapiens |B7Z5E3|B7Z5E3_HUMAN GN=LDHA PE=1 SV=2

Histone H2B type 1-O sp|P23527|H2B1O_HUMAN 3 23.00% OS=Homo sapiens GN=HIST1H2BO PE=1 SV=3

60S ribosomal protein L29 sp|P47914|RL29_HUMAN 2 10.10% OS=Homo sapiens GN=RPL29 PE=1 SV=2

Actin, cytoplasmic 1 sp|P60709|ACTB_HUMAN,sp 7 15.50% OS=Homo sapiens |P63261|ACTG_HUMAN GN=ACTB PE=1 SV=1 37

Table 3.1: Proteins identified in the detergent-insoluble portion of Kg1a cell lysate.

KG1a Np40 Lysis Mass Lysate Lysate Mass Spec Supernatant Pellet Spec Analysis Analysis Acetone precipitation Dissolve in Dissolve in urea/thiourea urea/thiourea Buffer Buffer

Mass Mass Tio2 Offgel Offgel Tio2 Spec fractionation fractionation enrichment Spec Analysis enrichment Analysis

Tio2 Tio2 enrichment enrichment

Mass Mass Spec Spec Analysis Analysis

Figure 3.2: Flow diagram of the Phosphoproteomics platform applied, a workflow scheme of different lysate samples ran on the orbitrap MS/MS for protein identification and Phosphorylation detection.

3.3 Enrichment Efficiency

The mass spectrometery results ascertain the importance of enrichment for phosphopeptides. The results generated from cell lysate directly without any enrichment when tested for phosphoproteins gave only 4 phosphoproteins with all phosphorylation occurring on a Tyrosine residue

(Appendix Table 1) while results generated after off-gel fractionation followed by Ti-O2 enrichment (using TiO2 micro columns kit (Protea) provided a list of 462 phosphoproteins (Appendix Table 2).

3.4 Phosphorylated proteins found in Kg1a cells

The identified 465 phosphorylated proteins, were mostly singly or

doubly phosphorylated (Figure 3.3). They were composed of 424

phosphorylated serine (pSer) residues, 1 phosphorylated threonine

(pThr) and 136 phosphorylated tyrosine (pTyr) sites (Figure 3.4).

Phosphorylation status

14.47 % 10.58 % 37.15 % single double triple 37.80 % > triple

Figure 3.3 Proportion of singly and multiply phosphorylated peptides in Kg1a cells.

Phosphorylation Distribution

0.18%

24.24%

Phosphorylation (S) Phosphorylation (T) 75.58% Phosphorylation (Y)

Figure 3.4 Distribution of identified phosphorylated serine, threonine, and tyrosine residues in resting Kg1a cells.

The phosphorylated proteins identified (462) were classified using gene ontology (Gene et al., 2011) for biological processes and found to span a wide range of categories (Appendix Table 3) of which regulation of transcription comprised the largest portion. Figure 3.5 shows phosphoproteins that are involved in different signaling pathways, including MAPKKK cascade and JNK cascade.

Selected GO classification of phosphoproteins intracellular receptor-mediated JNK cascade, 4signaling pathway, 4

regulatio n of regulation of protein MAPKKK cascade, 13 transcription, 30 kinase cascade, 6 hemopoiesis, 6

intracellular signaling cascade, 19

phosphorylation, 15

cell proliferation, 8

Figure 3.5 Frequency of phosphorylated proteins identified with selected GO biological process in Kg1a cells.

Interferon, gamma-inducible protein 16 (Ifi-16) is an example of one protein identified in our phosphoproteomic analysis of kg1a cells. Ifi-16 is involved in hematopoiesis, cell proliferation and regulation of transcription.

It is also known as interferon-inducible myeloid differentiation transcriptional activator as It is found to be constitutively expressed in human lymphoid cells and in cell lines of both the T and B lineages, But absent. In myeloid cells (HL-60, U937, K562) however, Ifi-16 is absent but is strongly induced in response to IFN-gamma.(Trapani et al., 1992) It is present in myeloid precursors (CD34+)+; such as the Kg1a cells analyzed here) and throughout monocyte development, but its expression is down- regulated in erythroid and polymorphonuclear precursor cells. It has been reported that it plays a role in the regulation of cell senescence and found in prostate, breast and ovarian cells.(Xin, Curry, Johnstone, Nickoloff, &

Choubey, 2003) It is also known to interact with cell cycle regulatory factors such as p53 and retinoblastoma-1., modulating p53 function, and inhibiting cell growth in the Ras/Raf signaling pathway.(Dawson, Elwood, Johnstone,

& Trapani, 1998) Thereby monitoring Ifi-16’s phosphorylation status before and after treatment would possibly reveal dynamics of the MAPK/ERK pathway. 42

Another relevant protein that was identified as phosphorylated in this cell extract is the leukocyte common antigen, CD45, also known as

Receptor-type tyrosine-protein phosphatase C. It’s involvement in various biological processes hints that its activation/deactivation mapping would be insightful. It has been characterized as an abundant transmembrane tyrosine phosphatase, expressed on all leukocytes, and is required for efficient lymphocyte signaling.(Tchilian et al., 2001). Deletion or mutations has been recognized as clinically relevant possibly causing SCID in mice. It has also been known to regulate Src family signaling by constitutive dephosphorylation in macrophages.(Roach et al., 1997) Recent evidence has supported its involvement in the regulation of hematopoietic stem cell migration. Studies on SCID mice showed impaired motility of CD34+ cells by inhibiting CD45 which also inhibited development of the hematopoietic progenitors revealing it’s central role in hematopoiesis as well as migration.(Shivtiel et al., 2011)

Integrin-linked kinase is another phophoprotein found in this cell line that is known to be involved in cell proliferation and signal transduction. A recent study on lung cancer cell line has reported that its over-expression 43 could promote cell proliferation and inhibit apoptosis.(Hu, Chen, Chen, Liu,

& Zhang, 2012). It is known to be autophosphorylated on serine residues.

Disruption of the interactions of ILK has been shown to eradicate leukemia in the bone marrow microenvironment by simultaneous targeting of both leukemic cells and activated bone marrow stromal cells. It represents a potential novel therapeutic strategy.(Tabe et al., 2007)

Quantitative characterization of phosphorylation dynamics of such influential proteins in response to different treatments is thought to shed light on details of cellular signaling networks that would set a platform for potential therapeutic targets.

3.5 Phosphorylated Proteins found in HL60 cells

Mass spectrometric analysis of HL60 lysate fractions identified 318 phosphoproteins .Gene ontology classified the identified proteins spanning

157 different GO classes , ranging from hematopoiesis, cell cycle and cell death regulation to differentiation. Figure 3.6 shows the distribution of proteins involved in differentiation and hematopoiesis. The 44 phosphoproteins identified comprise more than 50 involved in transcription. Studying the phosphorylation changes in these proteins promises to give insight into signaling pathways critical to these phenomena.

GO classification of selected phosphoproteins regulation of T cell differentiation monocyte 8% B cell differentiationdifferentiation 4% 8% hemopoiesis erythrocyte 29% differentiation 8%

regulation of leukocyte differentiation neutrophil myeloid cell 15% differentiation differentiation 4% 12% lymphocyte differentiation 12%

Figure 3.6 Frequency of phosphoproteins identified in HL60 cell-line involved in differentiation and hemopoiesis.

4. Discussion

The main objective of this study is to use quantitative proteomics to reveal the molecular effectors behind the blockage of differentiation and bring forth new therapeutic candidates for a targeted therapy for AML. In this preliminary work, a workflow of generating, processing and analyzing phosphoproteomic information from whole cell lysates has been achieved.

It is well known that mass spectrometery detects the most abundant proteins on the expense of rare or low concentration populations.

Phosphorylated proteins fall in the latter category (1-10% of the total proteome) despite their great impact in leading signaling pathways and impact on end states. This substochiometeric nature of phosphoproteins calls for a compilation of different techniques each revealing a part of the widely spread phosphoproteome.

A logical conclusion is the importance of enrichment which is strongly evidenced in this work by the big difference in the number of detected phosphoproteins with and without enrichment. Nevertheless extra phosphoprotein enrichment steps are intended for the following experimentation processes involving quantification and different time 46 points to detect more sensitive signaling changes against high background from the vast proteome.

It is important to note that the enrichment part is a major challenge for the scientific community in general and thus optimizing this part is of critical importance for these studies to take place and for relevant data to be extracted. Phosphopeptides also have multiple charge stages that have an effect on the enrichment methods of choice. Since there is no one single optimal strategy for phosphopeptide enrichment, we have decided to utilize a combination of strategies including TiO2, IMAC and immunoprecipitation using pTyr antibodies. In this preliminary experimentation only one enrichment strategy namely, TiO2 was used.

Without prior fractionation, there was no improved efficiency in detecting phosphopeptides (results not shown) but when off-gel isoelectric point based fractionation was employed, a significant improvement was observed highlighting the significance of these protocols on the efficiency of enrichment steps. The offgel fractionators is a system that fractionates proteins or peptides based on their isoelectric points (pI) in liquid phase. It provides additional sensitivity of downstream analysis tool such as HPLC 47 coupled with mass spectrometery. In addition, the pI is a useful parameter that can be used for validation of peptide MS hits. Other fractionation techniques like strong ion exchange chromatography (SCX) which hold promising effectiveness as a first dimension of separation are also intended to be used.

Implementation of bioinformatics tools is an integral part of this workflow; in this study Scaffold PTM program was used as a data filter for identification of phosphopeptides, and assignment of phosphorylation sites. It uses a probability based scoring system for phosporylation site assignment. For analysis of phosphorylated proteins identified we used another major bioinformatic tool, gene ontology (GO) enrichment, and were able to classify these proteins according to their relation to the different biological processes. Phosphorylation regulation could also be characterized using different clustering programs on the data generated from Mass spectrometry.

To profile the phosphorylation events during CD44-ligation induced differentiation, we aim to combine SILAC with strong cation exchange (SCX) 48 fractionation or/and off -gel isoelectric fractionation, TiO2 phosphopeptide enrichment, and high-accuracy and high-resolution liquid chromatography– tandem mass spectrometry (LC-MS/MS).

This thesis presents a minor, yet critical, step of a large project that aims to utilize knowledge about molecular biology of cancer and stem cell therapy in combination with technical expertise in phosphoproteomics to reveal crucial yet illusive biological pathways and signal transduction schemes involved in the pathology of Cancer. Confirmation of the identified pathways will need to be performed using Western blotting techniques and followed by loss of function or gain of function experiments to fully determine each pathways role in proliferation, differentiation induction and/or apoptosis. Although much work and challenges are anticipated, the aim of providing information that would help in generating more appropriate and customized therapeutic strategies for AML in specific and cancer in general is the awaited outcome.

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APPENDICES

Table 1: List of phosphorylated proteins identified without enrichment

Protein Name Accession Scaffold: Sequence Phosphoryla Phosphor Phosphor Protein Coverage tion (S) ylation ylation Probabili (T) (Y) ty

Transformer-2 protein sp|Q1359 0.9908 0.1206 1 homolog alpha 5|TRA2A_ OS=Homo sapiens HUMAN GN=TRA2A PE=1 SV=1 +2 +2

Uncharacterized tr|B4DQI6 0.9908 0.1206 1 protein OS=Homo |B4DQI6_ sapiens GN=TRA2A HUMAN PE=2 SV=1

Transformer-2 protein sp|Q1359 0.9908 0.1206 1 homolog alpha 5|TRA2A_ OS=Homo sapiens HUMAN GN=TRA2A PE=1 SV=1

Uncharacterized tr|B4DUA9 0.9908 0.1206 1 protein OS=Homo |B4DUA9_ sapiens GN=TRA2A HUMAN PE=2 SV=1

Table 2: List of Phosphorylated Proteins

Protein Name Serine(S) Tyrosine (T) Threonine (Y)

RNA-binding motif protein, Y chromosome, family 1 member B OS=Homo 3 1 sapiens GN=RBMY1B PE=2 SV=2 +5

RNA-binding motif protein, Y chromosome, family 1 member B OS=Homo 3 1 sapiens GN=RBMY1B PE=2 SV=2

Uncharacterized protein OS=Homo sapiens GN=SLTM PE=2 SV=1 3

Myosin-Ig OS=Homo sapiens GN=MYO1G PE=1 SV=2 1

Uncharacterized protein OS=Homo sapiens GN=DDX3X PE=2 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=EVL PE=2 SV=1 5

Uncharacterized protein OS=Homo sapiens GN=CLINT1 PE=2 SV=1 1 1

Uncharacterized protein OS=Homo sapiens GN=SAFB PE=2 SV=1 2

Uncharacterized protein OS=Homo sapiens GN=ACIN1 PE=4 SV=2 2 1

Uncharacterized protein OS=Homo sapiens GN=KHDRBS1 PE=4 SV=2 5

Uncharacterized protein OS=Homo sapiens GN=LSP1 PE=4 SV=1 2

Uncharacterized protein OS=Homo sapiens GN=EEF1D PE=3 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=EVL PE=4 SV=1 5

Uncharacterized protein OS=Homo sapiens GN=SAFB PE=4 SV=1 2

Uncharacterized protein OS=Homo sapiens GN=MCM2 PE=3 SV=1 3

Uncharacterized protein OS=Homo sapiens GN=SAFB PE=4 SV=1 2

ATP-dependent RNA helicase DDX3X OS=Homo sapiens GN=DDX3X PE=1 1 SV=3 +2

ATP-dependent RNA helicase DDX3X OS=Homo sapiens GN=DDX3X PE=1 1 SV=3 54

Core histone macro-H2A.1 OS=Homo sapiens GN=H2AFY PE=1 SV=4 1

Isoform 1 of Core histone macro-H2A.1 OS=Homo sapiens GN=H2AFY +2 1

Isoform 1 of Core histone macro-H2A.1 OS=Homo sapiens GN=H2AFY 1

Isoform 3 of Core histone macro-H2A.1 OS=Homo sapiens GN=H2AFY 1

Serine/arginine-rich splicing factor 10 OS=Homo sapiens GN=SRSF10 PE=1 1 SV=1

Isoform 2 of Serine/arginine-rich splicing factor 10 OS=Homo sapiens 1 GN=SRSF10 +1

Isoform 2 of Serine/arginine-rich splicing factor 10 OS=Homo sapiens 1 GN=SRSF10

Lupus La protein OS=Homo sapiens GN=SSB PE=1 SV=2 1

Nucleophosmin OS=Homo sapiens GN=NPM1 PE=1 SV=2 1

Isoform 2 of Nucleophosmin OS=Homo sapiens GN=NPM1 +1 1

Isoform 2 of Nucleophosmin OS=Homo sapiens GN=NPM1 1

RNA-binding motif protein, Y chromosome, family 1 member D OS=Homo 3 1 sapiens GN=RBMY1D PE=2 SV=1

Histone H1.4 OS=Homo sapiens GN=HIST1H1E PE=1 SV=2 +2 1

Histone H1.4 OS=Homo sapiens GN=HIST1H1E PE=1 SV=2 1

Inosine-5'-monophosphate dehydrogenase 2 OS=Homo sapiens 1 GN=IMPDH2 PE=1 SV=2

Stathmin OS=Homo sapiens GN=STMN1 PE=1 SV=3 1

Isoform 2 of Stathmin OS=Homo sapiens GN=STMN1 +1 1

Isoform 2 of Stathmin OS=Homo sapiens GN=STMN1 1

Nucleolin OS=Homo sapiens GN=NCL PE=1 SV=3 +1 1

Nucleolin OS=Homo sapiens GN=NCL PE=1 SV=3 1

Lamin-B1 OS=Homo sapiens GN=LMNB1 PE=1 SV=2 1

Heterogeneous nuclear ribonucleoproteins A2/B1 OS=Homo sapiens 5 1 GN=HNRNPA2B1 PE=1 SV=2 55

Isoform PML-5 of Protein PML OS=Homo sapiens GN=PML 3

Elongation factor 1-delta OS=Homo sapiens GN=EEF1D PE=1 SV=5 1

Isoform 2 of Elongation factor 1-delta OS=Homo sapiens GN=EEF1D +7 1

Isoform 2 of Elongation factor 1-delta OS=Homo sapiens GN=EEF1D 1

60S ribosomal protein L12 OS=Homo sapiens GN=RPL12 PE=1 SV=1 1

Lymphocyte-specific protein 1 OS=Homo sapiens GN=LSP1 PE=1 SV=1 +2 2

Lymphocyte-specific protein 1 OS=Homo sapiens GN=LSP1 PE=1 SV=1 2

Heterogeneous nuclear ribonucleoprotein G OS=Homo sapiens GN=RBMX 2 PE=1 SV=3 +1

Heterogeneous nuclear ribonucleoprotein G OS=Homo sapiens GN=RBMX 2 PE=1 SV=3

Lamina-associated polypeptide 2, isoforms beta/gamma OS=Homo 1 sapiens GN=TMPO PE=1 SV=2

DNA replication licensing factor MCM2 OS=Homo sapiens GN=MCM2 PE=1 3 SV=4 +1

DNA replication licensing factor MCM2 OS=Homo sapiens GN=MCM2 PE=1 3 SV=4

Heterogeneous nuclear ribonucleoprotein A3 OS=Homo sapiens 2 GN=HNRNPA3 PE=1 SV=2

Heterogeneous nuclear ribonucleoprotein M OS=Homo sapiens 3 GN=HNRNPM PE=1 SV=3

Isoform 2 of Heterogeneous nuclear ribonucleoprotein M OS=Homo 3 sapiens GN=HNRNPM +1

Isoform 2 of Heterogeneous nuclear ribonucleoprotein M OS=Homo 3 sapiens GN=HNRNPM

Triosephosphate isomerase OS=Homo sapiens GN=TPI1 PE=1 SV=3 1

Isoform 2 of Triosephosphate isomerase OS=Homo sapiens GN=TPI1 +1 1

Isoform 2 of Triosephosphate isomerase OS=Homo sapiens GN=TPI1 1

Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1 +3 1

Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1 1 56

Actin, cytoplasmic 2 OS=Homo sapiens GN=ACTG1 PE=1 SV=1 1

Spectrin beta chain, brain 1 OS=Homo sapiens GN=SPTBN1 PE=1 SV=2 2

Lamin-B2 OS=Homo sapiens GN=LMNB2 PE=1 SV=3 2

KH domain-containing, RNA-binding, signal transduction-associated 5 protein 1 OS=Homo sapiens GN=KHDRBS1 PE=1 SV=1

Isoform 2 of KH domain-containing, RNA-binding, signal transduction- 5 associated protein 1 OS=Homo sapiens GN=KHDRBS1 +3

Isoform 2 of KH domain-containing, RNA-binding, signal transduction- 5 associated protein 1 OS=Homo sapiens GN=KHDRBS1

Serine/arginine-rich splicing factor 1 OS=Homo sapiens GN=SRSF1 PE=1 1 SV=2

Serine/arginine-rich splicing factor 9 OS=Homo sapiens GN=SRSF9 PE=1 2 SV=1

Transcription intermediary factor 1-beta OS=Homo sapiens GN=TRIM28 4 PE=1 SV=5

Isoform 2 of Transcription intermediary factor 1-beta OS=Homo sapiens 4 GN=TRIM28 +1

Isoform 2 of Transcription intermediary factor 1-beta OS=Homo sapiens 4 GN=TRIM28

SNW domain-containing protein 1 OS=Homo sapiens GN=SNW1 PE=1 3 SV=1

Isoform 2 of Spectrin alpha chain, brain OS=Homo sapiens GN=SPTAN1 1

Clathrin interactor 1 OS=Homo sapiens GN=CLINT1 PE=1 SV=1 1 1

Isoform 2 of Clathrin interactor 1 OS=Homo sapiens GN=CLINT1 +2 1 1

Isoform 2 of Clathrin interactor 1 OS=Homo sapiens GN=CLINT1 1 1

Nuclear mitotic apparatus protein 1 OS=Homo sapiens GN=NUMA1 PE=1 1 SV=2

Isoform 2 of Nuclear mitotic apparatus protein 1 OS=Homo sapiens 1 GN=NUMA1 +1

Isoform 2 of Nuclear mitotic apparatus protein 1 OS=Homo sapiens 1 GN=NUMA1 57

RNA-binding motif protein, Y chromosome, family 1 member A1/C 3 1 OS=Homo sapiens GN=RBMY1A1 PE=1 SV=1

Isoform 3 of RNA-binding motif protein, Y chromosome, family 1 member 3 1 A1/C OS=Homo sapiens GN=RBMY1A1

RNA-binding motif protein, Y chromosome, family 1 member F/J 3 1 OS=Homo sapiens GN=RBMY1F PE=2 SV=2

Scaffold attachment factor B1 OS=Homo sapiens GN=SAFB PE=1 SV=4 +3 2

Scaffold attachment factor B1 OS=Homo sapiens GN=SAFB PE=1 SV=4 2

Plasminogen activator inhibitor 1 RNA-binding protein OS=Homo sapiens 1 GN=SERBP1 PE=1 SV=2

Isoform 2 of Plasminogen activator inhibitor 1 RNA-binding protein 1 OS=Homo sapiens GN=SERBP1 +1

Isoform 2 of Plasminogen activator inhibitor 1 RNA-binding protein 1 OS=Homo sapiens GN=SERBP1

Isoform Short of TATA-binding protein-associated factor 2N OS=Homo sapiens GN=TAF15 +1

Histone H2B type 1-M OS=Homo sapiens GN=HIST1H2BM PE=1 SV=3 2

Pinin OS=Homo sapiens GN=PNN PE=1 SV=4 1

SAFB-like transcription modulator OS=Homo sapiens GN=SLTM PE=1 SV=2 3 +1

SAFB-like transcription modulator OS=Homo sapiens GN=SLTM PE=1 SV=2 3

Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 1 OS=Homo sapiens GN=PAG1 PE=1 SV=2

Ena/VASP-like protein OS=Homo sapiens GN=EVL PE=1 SV=2 5

Isoform 1 of Ena/VASP-like protein OS=Homo sapiens GN=EVL +3 5

Isoform 1 of Ena/VASP-like protein OS=Homo sapiens GN=EVL 5

Isoform 1 of RNA-binding protein Raly OS=Homo sapiens GN=RALY 2

Apoptotic chromatin condensation inducer in the nucleus OS=Homo 2 1 sapiens GN=ACIN1 PE=1 SV=2 +1

Apoptotic chromatin condensation inducer in the nucleus OS=Homo 2 1 58 sapiens GN=ACIN1 PE=1 SV=2

Uncharacterized protein OS=Homo sapiens GN=ACTB PE=2 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=ACTG1 PE=3 SV=1 1

Histone deacetylase 2 OS=Homo sapiens GN=HDAC2 PE=1 SV=2 +1 1

Histone deacetylase 2 OS=Homo sapiens GN=HDAC2 PE=1 SV=2 1

Histone deacetylase OS=Homo sapiens GN=HDAC2 PE=2 SV=1 1

Isoform 2 of Septin-2 OS=Homo sapiens GN=SEPT2 +2 1

Isoform 2 of Septin-2 OS=Homo sapiens GN=SEPT2 1

Septin-2 OS=Homo sapiens GN=SEPT2 PE=1 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=SEPT2 PE=3 SV=1 1

Serine/threonine-protein kinase 10 OS=Homo sapiens GN=STK10 PE=1 1 SV=1

BUD13 homolog OS=Homo sapiens GN=BUD13 PE=1 SV=1 1 1

Calcium-regulated heat stable protein 1 OS=Homo sapiens GN=CARHSP1 1 1 PE=1 SV=2

Uncharacterized protein OS=Homo sapiens GN=DDX3X PE=2 SV=1 1

Uncharacterized protein C14orf43 OS=Homo sapiens GN=C14orf43 PE=1 1 SV=2 +1

Uncharacterized protein OS=Homo sapiens GN=C14orf43 PE=4 SV=1 1

Uncharacterized protein C14orf43 OS=Homo sapiens GN=C14orf43 PE=1 1 SV=2

Microtubule-associated protein 1S OS=Homo sapiens GN=MAP1S PE=1 1 SV=2 +1

Microtubule-associated protein 1S OS=Homo sapiens GN=MAP1S PE=1 1 SV=2

Uncharacterized protein OS=Homo sapiens GN=MAP1S PE=2 SV=1 1

Isoform 2 of Zinc finger CCCH-type antiviral protein 1 OS=Homo sapiens 1 GN=ZC3HAV1 +3

Isoform 3 of Zinc finger CCCH-type antiviral protein 1 OS=Homo sapiens 1 59

GN=ZC3HAV1

Uncharacterized protein OS=Homo sapiens GN=ZC3HAV1 PE=4 SV=1 1

Isoform 2 of Zinc finger CCCH-type antiviral protein 1 OS=Homo sapiens 1 GN=ZC3HAV1

Zinc finger CCCH-type antiviral protein 1 OS=Homo sapiens GN=ZC3HAV1 1 PE=1 SV=3

Histone H1.3 OS=Homo sapiens GN=HIST1H1D PE=1 SV=2 1

Histone H1.2 OS=Homo sapiens GN=HIST1H1C PE=1 SV=2 1

Phosphatidylserine synthase 2 OS=Homo sapiens GN=PTDSS2 PE=1 SV=1 1 +2

Phosphatidylserine synthase 2 OS=Homo sapiens GN=PTDSS2 PE=1 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=PTDSS2 PE=4 SV=1 1

Isoform 2 of AT-rich interactive domain-containing protein 1B OS=Homo 1 1 sapiens GN=ARID1B +2

Isoform 3 of AT-rich interactive domain-containing protein 1B OS=Homo 1 1 sapiens GN=ARID1B

Isoform 2 of AT-rich interactive domain-containing protein 1B OS=Homo 1 1 sapiens GN=ARID1B

AT-rich interactive domain-containing protein 1B OS=Homo sapiens 1 1 GN=ARID1B PE=1 SV=2

Isoform 2 of 6-phosphofructokinase, liver type OS=Homo sapiens 1 GN=PFKL +2

6-phosphofructokinase, liver type OS=Homo sapiens GN=PFKL PE=1 SV=6 1

Isoform 2 of 6-phosphofructokinase, liver type OS=Homo sapiens 1 GN=PFKL

Uncharacterized protein OS=Homo sapiens GN=PFKL PE=4 SV=1 1

Isoform Beta of E3 ubiquitin-protein ligase TRIM33 OS=Homo sapiens 1 GN=TRIM33 +2

Isoform Beta of E3 ubiquitin-protein ligase TRIM33 OS=Homo sapiens 1 60

GN=TRIM33

Uncharacterized protein OS=Homo sapiens GN=TRIM33 PE=4 SV=1 1

E3 ubiquitin-protein ligase TRIM33 OS=Homo sapiens GN=TRIM33 PE=1 1 SV=3

Transformer-2 protein homolog beta OS=Homo sapiens GN=TRA2B PE=1 1 SV=1 +1

Uncharacterized protein OS=Homo sapiens GN=TRA2B PE=4 SV=1 1

Transformer-2 protein homolog beta OS=Homo sapiens GN=TRA2B PE=1 1 SV=1

Isoform 2 of SH3 domain-containing kinase-binding protein 1 OS=Homo 1 sapiens GN=SH3KBP1 +4

Uncharacterized protein OS=Homo sapiens GN=SH3KBP1 PE=4 SV=1 1

Isoform 2 of SH3 domain-containing kinase-binding protein 1 OS=Homo 1 sapiens GN=SH3KBP1

SH3-domain kinase binding protein 1 OS=Homo sapiens GN=SH3KBP1 1 PE=2 SV=1

SH3 domain-containing kinase-binding protein 1 OS=Homo sapiens 1 GN=SH3KBP1 PE=1 SV=2

Uncharacterized protein OS=Homo sapiens GN=SH3KBP1 PE=2 SV=1 1

La-related protein 1 OS=Homo sapiens GN=LARP1 PE=1 SV=2 1 1

Uncharacterized protein OS=Homo sapiens GN=EEF1D PE=4 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=NCL PE=4 SV=1 1

DNA-directed RNA polymerase II subunit RPB1 OS=Homo sapiens 1 GN=POLR2A PE=1 SV=2

Metastasis-associated protein MTA1 OS=Homo sapiens GN=MTA1 PE=1 1 SV=2 +2

Metastasis-associated protein MTA1 OS=Homo sapiens GN=MTA1 PE=1 1 61

SV=2

Uncharacterized protein OS=Homo sapiens GN=MTA1 PE=4 SV=2 1

Uncharacterized protein OS=Homo sapiens GN=MTA1 PE=4 SV=1 1

RNA-binding protein 14 OS=Homo sapiens GN=RBM14 PE=1 SV=2 1 1

Isoform 2 of Receptor-type tyrosine-protein phosphatase C OS=Homo 1 sapiens GN=PTPRC +3

Uncharacterized protein OS=Homo sapiens GN=PTPRC PE=4 SV=1 1

Receptor-type tyrosine-protein phosphatase C OS=Homo sapiens 1 GN=PTPRC PE=1 SV=2

Isoform 2 of Receptor-type tyrosine-protein phosphatase C OS=Homo 1 sapiens GN=PTPRC

Isoform 2 of Protein PAT1 homolog 1 OS=Homo sapiens GN=PATL1 +2 1

Isoform 4 of Protein PAT1 homolog 1 OS=Homo sapiens GN=PATL1 1

Isoform 2 of Protein PAT1 homolog 1 OS=Homo sapiens GN=PATL1 1

Protein PAT1 homolog 1 OS=Homo sapiens GN=PATL1 PE=1 SV=2 1

Serine/threonine-protein kinase PAK 2 OS=Homo sapiens GN=PAK2 PE=1 1 SV=3

Antigen KI-67 OS=Homo sapiens GN=MKI67 PE=1 SV=2 +1 1

Antigen KI-67 OS=Homo sapiens GN=MKI67 PE=1 SV=2 1

Uncharacterized protein OS=Homo sapiens GN=MKI67 PE=4 SV=1 1

Ribosomal L1 domain-containing protein 1 OS=Homo sapiens GN=RSL1D1 1 PE=1 SV=3 +1

Uncharacterized protein OS=Homo sapiens GN=RSL1D1 PE=2 SV=1 1

Ribosomal L1 domain-containing protein 1 OS=Homo sapiens GN=RSL1D1 1 PE=1 SV=3

Drebrin-like protein OS=Homo sapiens GN=DBNL PE=1 SV=1 +2 1 1

Uncharacterized protein OS=Homo sapiens GN=DBNL PE=2 SV=1 1 1 62

Drebrin-like protein OS=Homo sapiens GN=DBNL PE=1 SV=1 1 1

Uncharacterized protein OS=Homo sapiens GN=DBNL PE=2 SV=1 1 1

Isoform Short of Myosin-IXb OS=Homo sapiens GN=MYO9B +1 1

Isoform Short of Myosin-IXb OS=Homo sapiens GN=MYO9B 1

Myosin-IXb OS=Homo sapiens GN=MYO9B PE=1 SV=3 1

Protein NDRG1 OS=Homo sapiens GN=NDRG1 PE=1 SV=1 +7 1 1

Uncharacterized protein OS=Homo sapiens GN=NDRG1 PE=4 SV=2 1 1

N-myc downstream regulated gene 1, isoform CRA_a OS=Homo sapiens 1 1 GN=NDRG1 PE=2 SV=1

Uncharacterized protein OS=Homo sapiens GN=NDRG1 PE=4 SV=1 1 1

Uncharacterized protein OS=Homo sapiens GN=NDRG1 PE=2 SV=1 1 1

Protein NDRG1 OS=Homo sapiens GN=NDRG1 PE=1 SV=1 1 1

Uncharacterized protein OS=Homo sapiens GN=NDRG1 PE=4 SV=1 1 1

Serine/threonine-protein kinase TAO3 OS=Homo sapiens GN=TAOK3 PE=1 1 SV=2 +1

Uncharacterized protein OS=Homo sapiens GN=TAOK3 PE=4 SV=1 1

Serine/threonine-protein kinase TAO3 OS=Homo sapiens GN=TAOK3 PE=1 1 SV=2

Isoform B of Eukaryotic translation initiation factor 4 gamma 1 OS=Homo 1 sapiens GN=EIF4G1 +8

Uncharacterized protein OS=Homo sapiens GN=EIF4G1 PE=4 SV=1 1

Isoform C of Eukaryotic translation initiation factor 4 gamma 1 OS=Homo 1 sapiens GN=EIF4G1

Eukaryotic translation initiation factor 4 gamma 1 OS=Homo sapiens 1 GN=EIF4G1 PE=1 SV=4

Isoform B of Eukaryotic translation initiation factor 4 gamma 1 OS=Homo 1 sapiens GN=EIF4G1 63

Isoform E of Eukaryotic translation initiation factor 4 gamma 1 OS=Homo 1 sapiens GN=EIF4G1

Uncharacterized protein OS=Homo sapiens GN=EIF4G1 PE=4 SV=1 1

Isoform D of Eukaryotic translation initiation factor 4 gamma 1 OS=Homo 1 sapiens GN=EIF4G1

Zinc finger CCCH domain-containing protein 4 OS=Homo sapiens 1 GN=ZC3H4 PE=1 SV=3

FK506-binding protein 15 OS=Homo sapiens GN=FKBP15 PE=1 SV=2 1

Probable ATP-dependent RNA helicase DDX10 OS=Homo sapiens 1 GN=DDX10 PE=1 SV=2 +1

Probable ATP-dependent RNA helicase DDX10 OS=Homo sapiens 1 GN=DDX10 PE=1 SV=2

Uncharacterized protein OS=Homo sapiens GN=DDX10 PE=4 SV=1 1

Isoform 2 of Zinc finger protein 638 OS=Homo sapiens GN=ZNF638 +5 1

Zinc finger protein 638 OS=Homo sapiens GN=ZNF638 PE=1 SV=2 1

Uncharacterized protein OS=Homo sapiens GN=ZNF638 PE=4 SV=1 1

Isoform 2 of Zinc finger protein 638 OS=Homo sapiens GN=ZNF638 1

Isoform 4 of Zinc finger protein 638 OS=Homo sapiens GN=ZNF638 1

Uncharacterized protein OS=Homo sapiens GN=ZNF638 PE=4 SV=1 1

Isoform 3 of Zinc finger protein 638 OS=Homo sapiens GN=ZNF638 1

Isoform 2 of Arf-GAP domain and FG repeats-containing protein 1 1 OS=Homo sapiens GN=AGFG1 +6

Uncharacterized protein OS=Homo sapiens GN=AGFG1 PE=4 SV=1 1

Arf-GAP domain and FG repeats-containing protein 1 OS=Homo sapiens 1 GN=AGFG1 PE=1 SV=2

Uncharacterized protein OS=Homo sapiens GN=AGFG1 PE=4 SV=1 1 64

Isoform 2 of Arf-GAP domain and FG repeats-containing protein 1 1 OS=Homo sapiens GN=AGFG1

Isoform 3 of Arf-GAP domain and FG repeats-containing protein 1 1 OS=Homo sapiens GN=AGFG1

Uncharacterized protein OS=Homo sapiens GN=AGFG1 PE=4 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=RBMX PE=2 SV=1 2

Heterogeneous nuclear ribonucleoprotein G-like 1 OS=Homo sapiens 2 GN=RBMXL1 PE=1 SV=1

Torsin-1A-interacting protein 1 OS=Homo sapiens GN=TOR1AIP1 PE=1 2 1 SV=2 +1

Uncharacterized protein OS=Homo sapiens GN=TOR1AIP1 PE=4 SV=1 2 1

Torsin-1A-interacting protein 1 OS=Homo sapiens GN=TOR1AIP1 PE=1 2 1 SV=2

ATP-citrate synthase OS=Homo sapiens GN=ACLY PE=1 SV=3 +1 2

Uncharacterized protein OS=Homo sapiens GN=ACLY PE=4 SV=2 2

ATP-citrate synthase OS=Homo sapiens GN=ACLY PE=1 SV=3 2

Pyruvate dehydrogenase E1 component subunit alpha, somatic form, 2 mitochondrial OS=Homo sapiens GN=PDHA1 PE=1 SV=3 +3

Pyruvate dehydrogenase E1 component subunit alpha, somatic form, 2 mitochondrial OS=Homo sapiens GN=PDHA1 PE=1 SV=3

Uncharacterized protein OS=Homo sapiens GN=PDHA1 PE=2 SV=1 2

Mitochondrial PDHA1 OS=Homo sapiens GN=PDHA1 PE=2 SV=1 2

Isoform 2 of RAF proto-oncogene serine/threonine-protein kinase 2 2 OS=Homo sapiens GN=RAF1 +3

Uncharacterized protein OS=Homo sapiens GN=RAF1 PE=2 SV=1 2 2

Isoform 2 of RAF proto-oncogene serine/threonine-protein kinase 2 2 OS=Homo sapiens GN=RAF1

RAF proto-oncogene serine/threonine-protein kinase OS=Homo sapiens 2 2 GN=RAF1 PE=1 SV=1 65

Uncharacterized protein OS=Homo sapiens GN=RAF1 PE=2 SV=1 2 2

Isoform 11 of Serine/threonine-protein kinase MARK2 OS=Homo sapiens 2 GN=MARK2 +8

Isoform 5 of Serine/threonine-protein kinase MARK2 OS=Homo sapiens 2 GN=MARK2

Serine/threonine-protein kinase MARK2 OS=Homo sapiens GN=MARK2 2 PE=1 SV=2

Isoform 4 of Serine/threonine-protein kinase MARK2 OS=Homo sapiens 2 GN=MARK2

Isoform 11 of Serine/threonine-protein kinase MARK2 OS=Homo sapiens 2 GN=MARK2

Isoform 9 of Serine/threonine-protein kinase MARK2 OS=Homo sapiens 2 GN=MARK2

Isoform 15 of Serine/threonine-protein kinase MARK2 OS=Homo sapiens 2 GN=MARK2

Isoform 8 of Serine/threonine-protein kinase MARK2 OS=Homo sapiens 2 GN=MARK2

Uncharacterized protein OS=Homo sapiens GN=MARK2 PE=4 SV=2 2

Isoform 16 of Serine/threonine-protein kinase MARK2 OS=Homo sapiens 2 GN=MARK2

Isoform 2 of B-cell lymphoma/leukemia 11A OS=Homo sapiens 2 GN=BCL11A +5

Isoform 6 of B-cell lymphoma/leukemia 11A OS=Homo sapiens 2 GN=BCL11A

Isoform 4 of B-cell lymphoma/leukemia 11A OS=Homo sapiens 2 GN=BCL11A

Uncharacterized protein OS=Homo sapiens GN=BCL11A PE=4 SV=1 2

B-cell lymphoma/leukemia 11A OS=Homo sapiens GN=BCL11A PE=1 SV=2 2

Isoform 2 of B-cell lymphoma/leukemia 11A OS=Homo sapiens 2 GN=BCL11A 66

Isoform 2 of YTH domain-containing protein 1 OS=Homo sapiens 2 GN=YTHDC1 +1

YTH domain-containing protein 1 OS=Homo sapiens GN=YTHDC1 PE=1 2 SV=3

Isoform 2 of YTH domain-containing protein 1 OS=Homo sapiens 2 GN=YTHDC1

Isoform 3 of Protein virilizer homolog OS=Homo sapiens GN=KIAA1429 +1 2

Isoform 3 of Protein virilizer homolog OS=Homo sapiens GN=KIAA1429 2

Protein virilizer homolog OS=Homo sapiens GN=KIAA1429 PE=1 SV=2 2

Secretory carrier-associated membrane protein 2 OS=Homo sapiens 2 GN=SCAMP2 PE=1 SV=2

Isoform 2 of DENN domain-containing protein 3 OS=Homo sapiens 2 GN=DENND3 +2

Isoform 2 of DENN domain-containing protein 3 OS=Homo sapiens 2 GN=DENND3

DENN domain-containing protein 3 OS=Homo sapiens GN=DENND3 PE=2 2 SV=2

Uncharacterized protein OS=Homo sapiens GN=DENND3 PE=4 SV=1 2

Isoform 3 of Rho GTPase-activating protein 25 OS=Homo sapiens 2 GN=ARHGAP25 +6

Uncharacterized protein OS=Homo sapiens GN=ARHGAP25 PE=4 SV=1 2

Isoform 4 of Rho GTPase-activating protein 25 OS=Homo sapiens 2 GN=ARHGAP25

Isoform 3 of Rho GTPase-activating protein 25 OS=Homo sapiens 2 GN=ARHGAP25

Rho GTPase-activating protein 25 OS=Homo sapiens GN=ARHGAP25 PE=1 2 SV=2

Uncharacterized protein OS=Homo sapiens GN=ARHGAP25 PE=4 SV=1 2

Uncharacterized protein OS=Homo sapiens GN=ARHGAP25 PE=4 SV=1 2 67

Isoform 2 of Gamma-interferon-inducible protein 16 OS=Homo sapiens 2 1 GN=IFI16 +2

Isoform 2 of Gamma-interferon-inducible protein 16 OS=Homo sapiens 2 1 GN=IFI16

Gamma-interferon-inducible protein 16 OS=Homo sapiens GN=IFI16 PE=1 2 1 SV=3

Isoform 3 of Gamma-interferon-inducible protein 16 OS=Homo sapiens 2 1 GN=IFI16

Centrosomal protein of 128 kDa OS=Homo sapiens GN=CEP128 PE=1 SV=2 2

Isoform 2 of Zinc finger CCCH domain-containing protein 13 OS=Homo 2 sapiens GN=ZC3H13 +1

Isoform 2 of Zinc finger CCCH domain-containing protein 13 OS=Homo 2 sapiens GN=ZC3H13

Zinc finger CCCH domain-containing protein 13 OS=Homo sapiens 2 GN=ZC3H13 PE=1 SV=1

Isoform 2 of PHD and RING finger domain-containing protein 1 OS=Homo 2 sapiens GN=PHRF1 +4

Uncharacterized protein OS=Homo sapiens GN=PHRF1 PE=4 SV=1 2

Isoform 2 of PHD and RING finger domain-containing protein 1 OS=Homo 2 sapiens GN=PHRF1

PHD and RING finger domain-containing protein 1 OS=Homo sapiens 2 GN=PHRF1 PE=1 SV=3

Isoform 3 of PHD and RING finger domain-containing protein 1 OS=Homo 2 sapiens GN=PHRF1

Uncharacterized protein OS=Homo sapiens GN=PHRF1 PE=4 SV=1 2

Isoform 2 of TRAF2 and NCK-interacting protein kinase OS=Homo sapiens 2 GN=TNIK +9

Isoform 2 of TRAF2 and NCK-interacting protein kinase OS=Homo sapiens 2 GN=TNIK

Isoform 4 of TRAF2 and NCK-interacting protein kinase OS=Homo sapiens 2 GN=TNIK

Isoform 8 of TRAF2 and NCK-interacting protein kinase OS=Homo sapiens 2 68

GN=TNIK

Isoform 5 of TRAF2 and NCK-interacting protein kinase OS=Homo sapiens 2 GN=TNIK

TRAF2 and NCK-interacting protein kinase OS=Homo sapiens GN=TNIK 2 PE=1 SV=1

Uncharacterized protein OS=Homo sapiens GN=TNIK PE=4 SV=1 2

Isoform 6 of TRAF2 and NCK-interacting protein kinase OS=Homo sapiens 2 GN=TNIK

Isoform 3 of TRAF2 and NCK-interacting protein kinase OS=Homo sapiens 2 GN=TNIK

Isoform 7 of TRAF2 and NCK-interacting protein kinase OS=Homo sapiens 2 GN=TNIK

Uncharacterized protein OS=Homo sapiens GN=TNIK PE=4 SV=1 2

Histone H2B type 1-K OS=Homo sapiens GN=HIST1H2BK PE=1 SV=3 +10 2

Histone H2B type 1-N OS=Homo sapiens GN=HIST1H2BN PE=1 SV=3 2

Histone H2B type 1-C/E/F/G/I OS=Homo sapiens GN=HIST1H2BC PE=1 2 SV=4

Histone H2B type 1-D OS=Homo sapiens GN=HIST1H2BD PE=1 SV=2 2

Histone H2B type 1-L OS=Homo sapiens GN=HIST1H2BL PE=1 SV=3 2

Histone H2B type F-S OS=Homo sapiens GN=H2BFS PE=1 SV=2 2

Histone H2B OS=Homo sapiens GN=HIST2H2BF PE=2 SV=1 2

Histone H2B type 1-K OS=Homo sapiens GN=HIST1H2BK PE=1 SV=3 2

Histone H2B type 1-H OS=Homo sapiens GN=HIST1H2BH PE=1 SV=3 2

Histone H2B type 2-F OS=Homo sapiens GN=HIST2H2BF PE=1 SV=3 2

Isoform 2 of Putative RNA-binding protein 15 OS=Homo sapiens 2 1 GN=RBM15 +2

Isoform 3 of Putative RNA-binding protein 15 OS=Homo sapiens 2 1 GN=RBM15 69

Isoform 2 of Putative RNA-binding protein 15 OS=Homo sapiens 2 1 GN=RBM15

Putative RNA-binding protein 15 OS=Homo sapiens GN=RBM15 PE=1 SV=2 2 1

Isoform 2 of Protein LSM14 homolog A OS=Homo sapiens GN=LSM14A +1 2 1

Protein LSM14 homolog A OS=Homo sapiens GN=LSM14A PE=1 SV=3 2 1

Isoform 2 of Protein LSM14 homolog A OS=Homo sapiens GN=LSM14A 2 1

Isoform 2 of Microtubule-associated protein 4 OS=Homo sapiens 2 1 GN=MAP4 +9

Microtubule-associated protein 4 OS=Homo sapiens GN=MAP4 PE=1 SV=3 2 1

Microtubule-associated protein OS=Homo sapiens GN=MAP4 PE=4 SV=1 2 1

Isoform 5 of Microtubule-associated protein 4 OS=Homo sapiens 2 1 GN=MAP4

Isoform 4 of Microtubule-associated protein 4 OS=Homo sapiens 2 1 GN=MAP4

Microtubule-associated protein OS=Homo sapiens GN=MAP4 PE=2 SV=1 2 1

Isoform 2 of Microtubule-associated protein 4 OS=Homo sapiens 2 1 GN=MAP4

Isoform 6 of Microtubule-associated protein 4 OS=Homo sapiens 2 1 GN=MAP4

Microtubule-associated protein OS=Homo sapiens GN=MAP4 PE=4 SV=1 2 1

Microtubule-associated protein OS=Homo sapiens GN=MAP4 PE=4 SV=3 2 1

Receptor expression-enhancing protein 4 OS=Homo sapiens GN=REEP4 2 PE=1 SV=1

Isoform 2 of Protein lunapark OS=Homo sapiens GN=LNP +5 2

Uncharacterized protein OS=Homo sapiens GN=KIAA1715 PE=4 SV=1 2

Isoform 2 of Protein lunapark OS=Homo sapiens GN=LNP 2

Isoform 3 of Protein lunapark OS=Homo sapiens GN=LNP 2

KIAA1715 protein OS=Homo sapiens GN=KIAA1715 PE=2 SV=1 2 70

Uncharacterized protein OS=Homo sapiens GN=KIAA1715 PE=4 SV=1 2

Protein lunapark OS=Homo sapiens GN=LNP PE=1 SV=2 2

Isoform 2 of Ataxin-2-like protein OS=Homo sapiens GN=ATXN2L +7 2

Isoform 5 of Ataxin-2-like protein OS=Homo sapiens GN=ATXN2L 2

Ataxin-2-like protein OS=Homo sapiens GN=ATXN2L PE=1 SV=2 2

Isoform 6 of Ataxin-2-like protein OS=Homo sapiens GN=ATXN2L 2

Isoform 2 of Ataxin-2-like protein OS=Homo sapiens GN=ATXN2L 2

Uncharacterized protein OS=Homo sapiens GN=ATXN2L PE=4 SV=1 2

Isoform 3 of Ataxin-2-like protein OS=Homo sapiens GN=ATXN2L 2

Isoform 4 of Ataxin-2-like protein OS=Homo sapiens GN=ATXN2L 2

Uncharacterized protein OS=Homo sapiens GN=ATXN2L PE=2 SV=1 2

Isoform 2 of CLK4-associating serine/arginine rich protein OS=Homo 2 sapiens GN=CLASRP +3

Uncharacterized protein OS=Homo sapiens GN=CLASRP PE=4 SV=1 2

Isoform 2 of CLK4-associating serine/arginine rich protein OS=Homo 2 sapiens GN=CLASRP

CLK4-associating serine/arginine rich protein OS=Homo sapiens 2 GN=CLASRP PE=1 SV=4

Uncharacterized protein OS=Homo sapiens GN=CLASRP PE=4 SV=1 2

Programmed cell death protein 4 OS=Homo sapiens GN=PDCD4 PE=1 SV=2 2 +1

Uncharacterized protein OS=Homo sapiens GN=PDCD4 PE=4 SV=2 2

Programmed cell death protein 4 OS=Homo sapiens GN=PDCD4 PE=1 SV=2 2

Isoform 3 of Alpha-adducin OS=Homo sapiens GN=ADD1 +1 2

Isoform 3 of Alpha-adducin OS=Homo sapiens GN=ADD1 2

Alpha-adducin OS=Homo sapiens GN=ADD1 PE=1 SV=2 2

Isoform 2 of Eukaryotic translation initiation factor 2A OS=Homo sapiens 2 GN=EIF2A +4 71

Uncharacterized protein OS=Homo sapiens GN=EIF2A PE=4 SV=1 2

Uncharacterized protein OS=Homo sapiens GN=EIF2A PE=2 SV=1 2

Isoform 2 of Eukaryotic translation initiation factor 2A OS=Homo sapiens 2 GN=EIF2A

Eukaryotic translation initiation factor 2A OS=Homo sapiens GN=EIF2A 2 PE=1 SV=3

Isoform 2 of Rho guanine nucleotide exchange factor 6 OS=Homo sapiens 2 GN=ARHGEF6 +2

Uncharacterized protein OS=Homo sapiens GN=ARHGEF6 PE=4 SV=1 2

Rho guanine nucleotide exchange factor 6 OS=Homo sapiens 2 GN=ARHGEF6 PE=1 SV=2

Isoform 2 of Rho guanine nucleotide exchange factor 6 OS=Homo sapiens 2 GN=ARHGEF6

Glycogen synthase kinase-3 alpha OS=Homo sapiens GN=GSK3A PE=1 2 SV=2

Isoform 2 of Bcl-2-associated transcription factor 1 OS=Homo sapiens 3 1 GN=BCLAF1 +1

Isoform 2 of Bcl-2-associated transcription factor 1 OS=Homo sapiens 3 1 GN=BCLAF1

Bcl-2-associated transcription factor 1 OS=Homo sapiens GN=BCLAF1 PE=1 3 1 SV=2

RNA-binding motif protein, Y chromosome, family 1 member E OS=Homo 3 1 sapiens GN=RBMY1E PE=2 SV=1

Isoform 3 of Cyclin-Y OS=Homo sapiens GN=CCNY +1 3 1

Isoform 3 of Cyclin-Y OS=Homo sapiens GN=CCNY 3 1

Cyclin-Y OS=Homo sapiens GN=CCNY PE=1 SV=2 3 1

Thyroid hormone receptor-associated protein 3 OS=Homo sapiens 3 GN=THRAP3 PE=1 SV=2

Isoform J of Kinesin light chain 1 OS=Homo sapiens GN=KLC1 +1 3 72

Isoform N of Kinesin light chain 1 OS=Homo sapiens GN=KLC1 3

Isoform J of Kinesin light chain 1 OS=Homo sapiens GN=KLC1 3

Isoform 3 of Histone deacetylase 7 OS=Homo sapiens GN=HDAC7 +6 3 1

Isoform 7 of Histone deacetylase 7 OS=Homo sapiens GN=HDAC7 3 1

Histone deacetylase 7 OS=Homo sapiens GN=HDAC7 PE=1 SV=2 3 1

Isoform 6 of Histone deacetylase 7 OS=Homo sapiens GN=HDAC7 3 1

Isoform 8 of Histone deacetylase 7 OS=Homo sapiens GN=HDAC7 3 1

Isoform 5 of Histone deacetylase 7 OS=Homo sapiens GN=HDAC7 3 1

Isoform 4 of Histone deacetylase 7 OS=Homo sapiens GN=HDAC7 3 1

Isoform 3 of Histone deacetylase 7 OS=Homo sapiens GN=HDAC7 3 1

Isoform 3 of Nuclear receptor corepressor 2 OS=Homo sapiens GN=NCOR2 3 1 +9

Uncharacterized protein OS=Homo sapiens GN=NCOR2 PE=4 SV=1 3 1

Uncharacterized protein OS=Homo sapiens GN=NCOR2 PE=4 SV=2 3 1

Isoform 3 of Nuclear receptor corepressor 2 OS=Homo sapiens GN=NCOR2 3 1

Isoform 4 of Nuclear receptor corepressor 2 OS=Homo sapiens GN=NCOR2 3 1

Uncharacterized protein OS=Homo sapiens GN=NCOR2 PE=4 SV=1 3 1

Isoform 5 of Nuclear receptor corepressor 2 OS=Homo sapiens GN=NCOR2 3 1

Uncharacterized protein OS=Homo sapiens GN=NCOR2 PE=4 SV=2 3 1

Uncharacterized protein OS=Homo sapiens GN=NCOR2 PE=4 SV=1 3 1

Nuclear receptor corepressor 2 OS=Homo sapiens GN=NCOR2 PE=1 SV=2 3 1

GTP-binding protein 1 OS=Homo sapiens GN=GTPBP1 PE=1 SV=3 +1 3

Uncharacterized protein OS=Homo sapiens GN=GTPBP1 PE=4 SV=1 3

GTP-binding protein 1 OS=Homo sapiens GN=GTPBP1 PE=1 SV=3 3

Zinc finger CCCH domain-containing protein 18 OS=Homo sapiens 3 GN=ZC3H18 PE=1 SV=2 +1 73

Zinc finger CCCH domain-containing protein 18 OS=Homo sapiens 3 GN=ZC3H18 PE=1 SV=2

Uncharacterized protein OS=Homo sapiens GN=ZC3H18 PE=4 SV=1 3

Isoform 2 of AT-rich interactive domain-containing protein 1A OS=Homo 4 sapiens GN=ARID1A +1

Isoform 2 of AT-rich interactive domain-containing protein 1A OS=Homo 4 sapiens GN=ARID1A

AT-rich interactive domain-containing protein 1A OS=Homo sapiens 4 GN=ARID1A PE=1 SV=3

Isoform 2 of RAS protein activator like-3 OS=Homo sapiens GN=RASAL3 +1 4 1

Isoform 2 of RAS protein activator like-3 OS=Homo sapiens GN=RASAL3 4 1

RAS protein activator like-3 OS=Homo sapiens GN=RASAL3 PE=1 SV=2 4 1

Kinesin light chain 2 OS=Homo sapiens GN=KLC2 PE=1 SV=1 +1 4

Kinesin light chain 2 OS=Homo sapiens GN=KLC2 PE=1 SV=1 4

Uncharacterized protein OS=Homo sapiens GN=KLC2 PE=2 SV=1 4

Isoform 3 of KH domain-containing, RNA-binding, signal transduction- 5 associated protein 1 OS=Homo sapiens GN=KHDRBS1

CapZ-interacting protein OS=Homo sapiens GN=RCSD1 PE=1 SV=1 +1 5 1

CapZ-interacting protein OS=Homo sapiens GN=RCSD1 PE=1 SV=1 5 1

Uncharacterized protein OS=Homo sapiens GN=RCSD1 PE=2 SV=1 5 1

Coiled-coil domain-containing protein 86 OS=Homo sapiens GN=CCDC86 5 PE=1 SV=1

Zinc finger protein 828 OS=Homo sapiens GN=ZNF828 PE=1 SV=2 6

Uncharacterized protein OS=Homo sapiens GN=SRRM1 PE=4 SV=2 8

Serine/arginine repetitive matrix protein 2 OS=Homo sapiens GN=SRRM2 12 4 PE=1 SV=2

GRB2-related adapter protein 2 OS=Homo sapiens GN=GRAP2 PE=1 SV=1 1 +5

Uncharacterized protein OS=Homo sapiens GN=GRAP2 PE=4 SV=1 1 74

Uncharacterized protein OS=Homo sapiens GN=GRAP2 PE=2 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=GRAP2 PE=4 SV=1 1

GRB2-related adapter protein 2 OS=Homo sapiens GN=GRAP2 PE=1 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=GRAP2 PE=2 SV=1 1

Dual specificity mitogen-activated protein kinase kinase 2 OS=Homo 1 sapiens GN=MAP2K2 PE=1 SV=1

Mediator of RNA polymerase II transcription subunit 1 OS=Homo sapiens 1 GN=MED1 PE=1 SV=4

Isoform 2 of AP2-associated protein kinase 1 OS=Homo sapiens GN=AAK1 1 +2

AP2-associated protein kinase 1 OS=Homo sapiens GN=AAK1 PE=1 SV=3 1

Isoform 2 of AP2-associated protein kinase 1 OS=Homo sapiens GN=AAK1 1

Uncharacterized protein OS=Homo sapiens GN=AAK1 PE=4 SV=1 1

Integrin-linked protein kinase OS=Homo sapiens GN=ILK PE=1 SV=2 1

Upstream stimulatory factor 1 OS=Homo sapiens GN=USF1 PE=1 SV=1 +2 1

Uncharacterized protein OS=Homo sapiens GN=USF1 PE=4 SV=1 1

Uncharacterized protein OS=Homo sapiens GN=usf1-bd PE=2 SV=1 1

Upstream stimulatory factor 1 OS=Homo sapiens GN=USF1 PE=1 SV=1 1

Isoform 2 of Mediator of DNA damage checkpoint protein 1 OS=Homo 2 sapiens GN=MDC1 +8

Isoform 2 of Mediator of DNA damage checkpoint protein 1 OS=Homo 2 sapiens GN=MDC1

Uncharacterized protein OS=Homo sapiens GN=MDC1 PE=4 SV=1 2

Isoform 3 of Mediator of DNA damage checkpoint protein 1 OS=Homo 2 sapiens GN=MDC1

Uncharacterized protein OS=Homo sapiens GN=MDC1 PE=4 SV=1 2

Uncharacterized protein OS=Homo sapiens GN=MDC1 PE=4 SV=1 2 75

Uncharacterized protein OS=Homo sapiens GN=MDC1 PE=4 SV=1 2

Mediator of DNA damage checkpoint protein 1 OS=Homo sapiens 2 GN=MDC1 PE=1 SV=3

Uncharacterized protein OS=Homo sapiens GN=MDC1 PE=4 SV=1 2

Isoform Alpha of Zinc finger protein GLI2 OS=Homo sapiens GN=GLI2 +4 2

Zinc finger protein GLI2 OS=Homo sapiens GN=GLI2 PE=1 SV=4 2

Isoform Gamma of Zinc finger protein GLI2 OS=Homo sapiens GN=GLI2 2

Isoform Delta of Zinc finger protein GLI2 OS=Homo sapiens GN=GLI2 2

Isoform Beta of Zinc finger protein GLI2 OS=Homo sapiens GN=GLI2 2

Isoform Alpha of Zinc finger protein GLI2 OS=Homo sapiens GN=GLI2 2

Table 3: Biological process GO terms of Phosphorylated proteins

Term Count % PValue

GO:0034728~nucleosome organization 16 11.76471 1.30E-15

GO:0006334~nucleosome assembly 15 11.02941 7.74E-15

GO:0031497~chromatin assembly 15 11.02941 1.30E-14

GO:0065004~protein-DNA complex assembly 15 11.02941 2.50E-14

GO:0016071~mRNA metabolic process 24 17.64706 4.28E-14

GO:0006323~DNA packaging 16 11.76471 4.61E-14

GO:0034621~cellular macromolecular complex subunit 23 16.91176 1.96E-13 organization

GO:0006397~mRNA processing 22 16.17647 2.31E-13

GO:0008380~RNA splicing 21 15.44118 2.32E-13

GO:0006333~chromatin assembly or disassembly 15 11.02941 2.82E-12

GO:0006396~RNA processing 25 18.38235 2.03E-11

GO:0043933~macromolecular complex subunit organization 28 20.58824 2.45E-11

GO:0006325~chromatin organization 21 15.44118 4.45E-11

GO:0034622~cellular macromolecular complex assembly 19 13.97059 1.55E-10

GO:0051276~chromosome organization 22 16.17647 5.60E-10

GO:0065003~macromolecular complex assembly 25 18.38235 1.11E-09

GO:0000375~RNA splicing, via transesterification reactions 11 8.088235 6.34E-07

GO:0000377~RNA splicing, via transesterification reactions with 11 8.088235 6.34E-07 bulged adenosine as nucleophile

GO:0000398~nuclear mRNA splicing, via spliceosome 11 8.088235 6.34E-07

GO:0016584~nucleosome positioning 3 2.205882 0.001033

GO:0045934~negative regulation of nucleobase, nucleoside, 13 9.558824 0.001257 77 nucleotide and nucleic acid metabolic process

GO:0051172~negative regulation of nitrogen compound metabolic 13 9.558824 0.001411 process

GO:0006468~protein amino acid phosphorylation 15 11.02941 0.001424

GO:0022618~ribonucleoprotein complex assembly 5 3.676471 0.002714

GO:0006376~mRNA splice site selection 3 2.205882 0.005996

GO:0016310~phosphorylation 15 11.02941 0.007276

GO:0010605~negative regulation of macromolecule metabolic 14 10.29412 0.008768 process

GO:0051348~negative regulation of transferase activity 5 3.676471 0.008771

GO:0007010~cytoskeleton organization 10 7.352941 0.011365

GO:0007265~Ras protein signal transduction 5 3.676471 0.011916

GO:0030518~steroid hormone receptor signaling pathway 4 2.941176 0.01285

GO:0007254~JNK cascade 4 2.941176 0.013457

GO:0043408~regulation of MAPKKK cascade 5 3.676471 0.013519

GO:0016481~negative regulation of transcription 10 7.352941 0.015459

GO:0031098~stress-activated protein kinase signaling pathway 4 2.941176 0.016046

GO:0007242~intracellular signaling cascade 19 13.97059 0.0175

GO:0032989~cellular component morphogenesis 9 6.617647 0.019001

GO:0045893~positive regulation of transcription, DNA-dependent 10 7.352941 0.019354

GO:0051254~positive regulation of RNA metabolic process 10 7.352941 0.020309

GO:0048025~negative regulation of nuclear mRNA splicing, via 2 1.470588 0.02507 spliceosome

GO:0030522~intracellular receptor-mediated signaling pathway 4 2.941176 0.025383

GO:0010629~negative regulation of gene expression 10 7.352941 0.026473

GO:0032583~regulation of gene-specific transcription 5 3.676471 0.026627 78

GO:0040029~regulation of gene expression, epigenetic 4 2.941176 0.027169

GO:0051493~regulation of cytoskeleton organization 5 3.676471 0.027917

GO:0000245~spliceosome assembly 3 2.205882 0.02962

GO:0007569~cell aging 3 2.205882 0.03136

GO:0010639~negative regulation of organelle organization 4 2.941176 0.031921

GO:0048096~chromatin-mediated maintenance of transcription 2 1.470588 0.033288

GO:0050686~negative regulation of mRNA processing 2 1.470588 0.033288

GO:0006796~phosphate metabolic process 15 11.02941 0.034016

GO:0006793~phosphorus metabolic process 15 11.02941 0.034016

GO:0010927~cellular component assembly involved in 3 2.205882 0.036815 morphogenesis

GO:0030521~androgen receptor signaling pathway 3 2.205882 0.036815

GO:0043193~positive regulation of gene-specific transcription 4 2.941176 0.037079

GO:0006469~negative regulation of protein kinase activity 4 2.941176 0.037079

GO:0045935~positive regulation of nucleobase, nucleoside, 11 8.088235 0.037692 nucleotide and nucleic acid metabolic process

GO:0033673~negative regulation of kinase activity 4 2.941176 0.040367

GO:0010558~negative regulation of macromolecule biosynthetic 10 7.352941 0.041369 process

GO:0033119~negative regulation of RNA splicing 2 1.470588 0.041437

GO:0010551~regulation of specific transcription from RNA 4 2.941176 0.044973 polymerase II promoter

GO:0051173~positive regulation of nitrogen compound metabolic 11 8.088235 0.045094 process

GO:0006405~RNA export from nucleus 3 2.205882 0.046641

GO:0010553~negative regulation of specific transcription from 3 2.205882 0.046641 RNA polymerase II promoter 79

GO:0043242~negative regulation of protein complex disassembly 3 2.205882 0.046641

GO:0031327~negative regulation of cellular biosynthetic process 10 7.352941 0.04725

GO:0045941~positive regulation of transcription 10 7.352941 0.04858

GO:0050657~nucleic acid transport 4 2.941176 0.048592

GO:0050658~RNA transport 4 2.941176 0.048592

GO:0051236~establishment of RNA localization 4 2.941176 0.048592

GO:0030097~hemopoiesis 6 4.411765 0.049315

GO:0006403~RNA localization 4 2.941176 0.052349

GO:0009890~negative regulation of biosynthetic process 10 7.352941 0.052717

GO:0080135~regulation of cellular response to stress 4 2.941176 0.056243

GO:0010628~positive regulation of gene expression 10 7.352941 0.056585

GO:0045815~positive regulation of gene expression, epigenetic 2 1.470588 0.057531

GO:0042921~glucocorticoid receptor signaling pathway 2 1.470588 0.057531

GO:0010627~regulation of protein kinase cascade 6 4.411765 0.059414

GO:0006096~glycolysis 3 2.205882 0.059532

GO:0045449~regulation of transcription 30 22.05882 0.061656

GO:0043410~positive regulation of MAPKKK cascade 3 2.205882 0.061787

GO:0032582~negative regulation of gene-specific transcription 3 2.205882 0.061787

GO:0031328~positive regulation of cellular biosynthetic process 11 8.088235 0.063242

GO:0031958~corticosteroid receptor signaling pathway 2 1.470588 0.065477

GO:0022613~ribonucleoprotein complex biogenesis 5 3.676471 0.065703

GO:0009891~positive regulation of biosynthetic process 11 8.088235 0.068299

GO:0043244~regulation of protein complex disassembly 3 2.205882 0.068724

GO:0048534~hemopoietic or lymphoid organ development 6 4.411765 0.068819 80

GO:0000165~MAPKKK cascade 5 3.676471 0.070026

GO:0015931~nucleobase, nucleoside, nucleotide and nucleic acid 4 2.941176 0.070179 transport

GO:0030705~cytoskeleton-dependent intracellular transport 3 2.205882 0.071092

GO:0045197~establishment or maintenance of epithelial cell 2 1.470588 0.073357 apical/basal polarity

GO:0008283~cell proliferation 8 5.882353 0.075394

GO:0051494~negative regulation of cytoskeleton organization 3 2.205882 0.078349

GO:0000902~cell morphogenesis 7 5.147059 0.080369

GO:0006338~chromatin remodeling 3 2.205882 0.080818

GO:0042255~ribosome assembly 2 1.470588 0.081171

GO:0016568~chromatin modification 6 4.411765 0.081918

GO:0010552~positive regulation of specific transcription from RNA 3 2.205882 0.083311 polymerase II promoter

GO:0002520~immune system development 6 4.411765 0.083891

GO:0051253~negative regulation of RNA metabolic process 7 5.147059 0.085476

GO:0006007~glucose catabolic process 3 2.205882 0.085828

GO:0030520~estrogen receptor signaling pathway 2 1.470588 0.088919

GO:0051168~nuclear export 3 2.205882 0.090928

GO:0045944~positive regulation of transcription from RNA 7 5.147059 0.093467 polymerase II promoter

GO:0051338~regulation of transferase activity 7 5.147059 0.094379

GO:0048024~regulation of nuclear mRNA splicing, via spliceosome 2 1.470588 0.096603