For Research Use Only. RN003P Not for use in diagnostic procedures. Page 1 of 4 RIP-Certified Antibody Anti-EIF4G2 Code No. Quantity Concentration Form RN003P 200 L 1 mg/mL Affinity Purified

BACKGROUND: The eukaryote polypeptide chain REACTIVITY: This antibody reacts with human eIF4G forms complex with the mRNA EIF4G2 (~102 kDa) on Western blotting, cap-binding eIF4E and the ATP dependent RNA Immunoprecipitation and RNP Immunoprecipitation. helicase eIF4A, constituting the initiation factor eIF4F complex. The eIF4G plays an important role in the APPLICATIONS: mechanism of by tethering between other RNP Immunoprecipitation; 15 g/500 L of cell extract components of the ribosomal initiation factors. Two forms from 4.5 x 106 cells of eIF4G have been reported as two different products, Western blotting; 1:1,000 for chemiluminescence detection eIF4G1 and 2. The eIF4G2 was identified as a functional system homologue of eIF4G1. The homology of eIF4G2 to eIF4G1 Immunoprecipitation; 5 g/250 L of cell extract from is particularly high in central and carboxyl portions which 2.5 x 106 cells corresponds to the eIF4A and eIF3 binding region. The Immunohistochemistry; Not tested proteolysis of both eIF4G1 and eIF4G2 is associated with a Immunocytochemistry; Not tested decrease in cell viability and the inhibition of protein Flow cytometry; Not tested synthesis. Detailed procedure is provided in the following RIP-CERTIFIED ANTIBODY: PROTOCOLS. Posttranscriptional regulation of gene expression is a ribonucleoprotein-driven process, which involves RNA INTENDED USE: binding (RBPs) and non-coding RNAs that affect For Research Use Only. Not for use in diagnostic procedures. splicing, nuclear export, subcellular localization, mRNA decay and translation. The RNP Immunoprecipitation-Chip (RIP-Chip), RIP-Seq and RIP-RTPCR allow the REFERENCES: identification of multiple RNA targets of RBPs globally 1) Morley, S. J., and Pain, V. M., FEBS Lett. 503, 206-212 (2001) and within the context of a cell extract. Antibodies specific 2) Gradi, A., et al., Mol. Cell Biol. 18, 334-342 (1998) to the RNA binding protein of interest are used to co-immunoprecipitate the RNA binding protein and the associated subset of mRNAs. The mRNA content is SPECIES CROSS REACTIVITY: interrogated using standard microarray or sequencing Species Human Mouse Rat Hamster technology. RIP-Certified Antibody is validated for use in MDA-MB-231 RNP Immunoprecipitation (RIP) in conjunction with the NIH/3T3, Cells K562, 293T, Rat1 CHO WR19L RIP-Assay Kit distributed from MBL. Its ability to HeLa, Jurkat immunoprecipitate mRNAs and RBPs complex was confirmed by quantitative and qualitative analysis on Reactivity on WB + + + + NanoDrop, Bioanalyzer and RT-PCR or microarray.

LICENSING OPPORTUNITY: The RIP-Assay uses SOURCE: This antibody was purified from rabbit serum patented technology (US patent No. 6,635,422, US patent by affinity column chromatography. The rabbit was No. 7,504,210) of Ribonomics, Inc. MBL manufactures and immunized with KLH conjugated synthetic peptide, distributes this product under license from Ribonomics, Inc. EVNPPSDETDSSSAPSKE corresponding to 783-800 aa. Researchers may use this product for their own research. Researchers are not allowed to use this product or FORMULATION: 200 L volume of PBS containing RIP-Assay technology for commercial purpose without a 50% glycerol, pH 7.2. No preservative is contained. license. For commercial use, please contact us for licensing opportunities at [email protected] STORAGE: This antibody solution is stable for one year from the date of purchase when stored at -20oC.

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL https://ruo.mbl.co.jp/je/rip-assay/ e-mail [email protected], TEL 052-238-1904 RN003P Page 2 of 4

Normal Rabbit IgG anti-EIF4G2 5) Mix both 25 L of 50% protein A agarose beads slurry resuspended in nuclease-free PBS and Normal Rabbit IgG (RIP-Assay Kit) or anti-EIF4G2 antibody at the amount of suggested in the APPLICATIONS, and then add 1 mL of Wash buffer (+) into each tube. Incubate with gently agitation for 1 hour at 4oC. RNA Intensity RNA 6) Wash the beads once with ice-cold Lysis Buffer (+) (centrifuge the tube at 2,000 x g for 1 minute). Carefully Nucleotide length discard the supernatant using a pipettor without disturbing

G the beads. g I

t i 7) Add 500 L of cell lysate (precleared sample of step 4), b o b 2 then incubate with gentle agitation for 3 hours at 4 C. a G A R 4 N r l F 8) Wash the beads 4 times with Wash Buffer (+) (centrifuge I R e E l - d ma i a the tube at 2,000 x g for 1 minute). d r t t a o n o L N a T 9) Add 400 L of Master mix solution (Solution I: Solution II = 10 L: 390 L). Vortex for 10 seconds. 28 S 10) Add 250 L of Solution III. Vortex for 10 seconds. 18 S 11) Centrifuge the tube at 2,000 x g for 2 minutes. 12) Transfer the supernatant to the tube containing 2 L of Solution IV. 13) Add 600 L of ice-cold 2-propanol and place at -20oC for 20 minutes. Centrifuge the tube at 12,000 x g for 10 minutes. Analysis of isolated RNA with Bioanalyzer. 14) Wash the pellet 2 times with 0.5 mL of ice-cold 70% Ethanol and dry up the pellet for 5-15 minutes. Average of the RNA Quantity (n=2) 15) Dissolve the pellets in nuclease-free water. Antibody RNA (ng) RNA was quantified with NanoDrop (Thermo Fisher Normal Rabbit IgG 26.0 Scientific Inc.) and the RNA quality was analyzed with Bioanalyzer (Agilent Technologies, Inc.). anti-EIF4G2 115.0

Toatal RNA 72075.0 (Positive control for RNP Immunoprecipitation; MDA-MB-231)

PROTOCOLS: RNP Immunoprecipitation kDa 12345678 Some buffer and reagents are included in the RIP-Assay Kit 150 (code. RN1001). Please also refer to the protocol packaged in the RIP-Assay Kit. 100 [Material Preparation] 1. Lysis Buffer (+) 75 Before using the Lysis Buffer, protease inhibitors, RNase inhibitors, and DTT are added to the Lysis Buffer at the appropriate concentration. 2. Wash Buffer (+) 50 Before using the Wash Buffer, DTT is added to the Wash Buffer at the appropriate concentration. Western blot analysis of EIF4G2 expression in K562 (1), 293T (2), HeLa (3), Protocol Jurkat (4), NIH/3T3 (5), WR19L (6) Rat1 (7) 1) Wash 4.5 x 106 cells 2 times with PBS and resuspend and CHO (8) using RN003P. them with 500 L of ice-cold Lysis Buffer (+) containing

appropriate protease inhibitors, RNase inhibitors, and SDS-PAGE & Western Blotting DTT. Vortex for 10 seconds. Leave on ice for 10 minutes. 7 2) Centrifuge the tube at 12,000 x g for 5 minutes at 4oC and 1) Wash 1 x 10 cells 3 times with PBS and suspend them in transfer the supernatant to another tube. 1 mL of Laemmli’s sample buffer. 3) Add 25 L of 50% protein A agarose beads slurry 2) Boil the samples for 2 minutes and centrifuge. Load 10 L resuspended in Lysis Buffer (+) into the supernatant. of the sample per lane in a 1 mm thick Incubate it at 4oC with rotating for 1 hour. SDS-polyacrylamide gel for electrophoresis. o 3) Blot the protein to a polyvinylidene difluoride (PVDF) 4) Centrifuge the tube at 2,000 x g for 1 minute at 4 C and 2 transfer the supernatant to another tube (precleared membrane at 1 mA/cm for 1 hour in a semi-dry transfer sample). system (Transfer Buffer: 25 mM Tris, 190 mM glycine,

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20% MeOH). See the manufacture's manual for precise sample). transfer procedure. 5) Mix both 20 L of 50% protein A agarose beads slurry 4) To reduce nonspecific binding, soak the membrane in 10% resuspended in nuclease-free PBS and Normal Rabbit IgG skimmed milk (in PBS, pH 7.2) for 1 hour at room (RIP-Assay Kit) or anti-EIF4G2 antibody at the amount of temperature, or overnight at 4oC. suggested in the APPLICATIONS, and then add 1 mL of 5) Incubate the membrane with primary antibody diluted Wash buffer into each tube. Incubate with gently agitation with PBS, pH 7.2 containing 1% skimmed milk as for 1 hour at 4oC. suggested in the APPLICATIONS for 1 hour at room 6) Wash the beads once with ice-cold Lysis Buffer temperature. (The concentration of antibody will depend (centrifuge the tube at 2,000 x g for 1 minute). Carefully on condition.) discard the supernatant using a pipettor without disturbing 6) Wash the membrane with PBS-T [0.05% Tween-20 in the beads. PBS] (5 minutes x 3 times). 7) Add 250 L of cell lysate (precleared sample of step 4), 7) Incubate the membrane with the 1:10,000 then incubate with gentle agitation for 1 hour at 4oC HRP-conjugated Anti-rabbit IgG serum (MBL; code no. 8) Wash the beads 4 times with Wash Buffer (centrifuge the 458) diluted with 1% skimmed milk (in PBS, pH 7.2) for tube at 2,000 x g for 1 minute). 1 hour at room temperature. 9) Resuspend the beads in 20 L of Laemmli’s sample buffer, 8) Wash the membrane with PBS-T (5 minutes x 3 times). boil for 3-5 minutes, and centrifuge for 5 minutes. Use 20 9) Wipe excess buffer on the membrane, then incubate it L/lane for the SDS-PAGE analysis. with appropriate chemiluminescence reagent for 1 minute. (See SDS-PAGE & Western blotting.) Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. (Positive control for Immunoprecipitation; MDA-MB-231) 10) Expose to an X-ray film in a dark room for 1 minute. Develop the film as usual. The condition for exposure and development may vary. RELATED PRODUCTS: RIP-Assay Kit (Positive controls for Western blotting; MBA-MB-231, RN1001 RIP-Assay Kit K562, 293T, HeLa, Jurkat, NIH/3T3, WR19L, Rat1, CHO) RIP Certified Antibody RN001P Anti-EIF4E (polyclonal) RN002P Anti-EIF4G1 (polyclonal) kDa 12 RN003P Anti-EIF4G2 (polyclonal) 150 RN004P Anti-ELAVL1/HuR (polyclonal) RN005P Anti-ELAVL2/HuB (polyclonal) RN006P Anti-ELAVL3/HuC (polyclonal) 100 EIF4G2 RN007P Anti-IGF2BP1/IMP1 (polyclonal) RN008P Anti-IGF2BP2/IMP2 (polyclonal) 75 RN009P Anti-IGF2BP3/IMP3 (polyclonal) RN010P Anti-MSI1/Musashi1 (polyclonal)

Other RIP-Certified Antibodies are also available. 50 IgG heavy chain Please visit our website at https://ruo.mbl.co.jp/je/rip-assay/

Immunoprecipitation of EIF4G2 from RIP-Assay Starter Kit MDA-MB-231 with normal rabbit IgG (1) Each RIP-Assay Starter Kit contains 40 g of RIP-Certified or RN003P (2). After immunoprecipitated Antibody and RIP-Assay Kit. with the antibody, immunocomplex was resolved on SDS-PAGE and immunoblotted RN001PK RIP-Assay Starter Kit EIF4E (polyclonal) with RN003P. RN002PK RIP-Assay Starter Kit EIF4G1 (polyclonal) RN003PK RIP-Assay Starter Kit EIF4G2 (polyclonal) Immunoprecipitation RN004PK RIP-Assay Starter Kit ELAVL1/HuR (polyclonal) 1) Wash 1 x 107 cells 2 times with PBS and resuspend them RN005PK RIP-Assay Starter Kit ELAVL2/HuB (polyclonal) with 1 mL of ice-cold Lysis buffer (RIP-Assay Kit) RN006PK RIP-Assay Starter Kit ELAVL3/HuC (polyclonal) containing appropriate protease inhibitors, then sonicate RN007PK RIP-Assay Starter Kit IGF2BP1/IMP1 (polyclonal) briefly (up to 10 seconds). RN008PK RIP-Assay Starter Kit IGF2BP2/IMP2 (polyclonal) 2) Centrifuge the tube at 12,000 x g for 5 minutes at 4oC and RN009PK RIP-Assay Starter Kit IGF2BP3/IMP3 (polyclonal) transfer the supernatant to another tube. RN010PK RIP-Assay Starter Kit MSI1/Musashi1 (polyclonal) 3) Add 20 L of 50% protein A agarose beads slurry resuspended in Lysis Buffer into the supernatant. Incubate Other RIP Starter Kits are also available. it at 4oC with rotating for 1 hour. Please visit our website at https://ruo.mbl.co.jp/je/rip-assay/ 4) Centrifuge the tube at 2,000 x g for 1 minute at 4oC and transfer the supernatant to another tube (precleared

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RBP Antibody RBP antibody works on WB and/or IP, but not certified for working on RIP-Assay.

RN023PW Anti-PABPN1 (polyclonal) RN028PW Anti-EIF2C1/AGO1 (polyclonal) RN029PW Anti-EIF2C2/AGO2 (polyclonal) RN030PW Anti-DICER1 (polyclonal) RN031PW Anti-ZFP36 (polyclonal) RN034PW Anti-CUGBP1 (polyclonal) RN035PW Anti-CUGBP2 (polyclonal) RN002MW Anti-CUGBP1 (3B1) RN003MW Anti-EIF2C2/AGO2 (1B1-E2H5)

100203-2.1

This product is manufactured and distributed by MBL under license from Ribonomics, Inc.