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To Download the Supplementary Data Of SUPPLEMENTARY INFORMATION doi:10.1038/nature10719 0 1 2 3 4 5 6 7 8 9 kb gag env 5′ LTR MA CA NC p6 pol vif nef tat vpu rev 3′ LTR pr rt int vpr gp120 gp41 0 1 2 3 Gag4 -SF5 6 Pol(-) 7 8-SF Vif -SF9 kb Gp160 -SF gag MA -SF PR(-)-SF Vpr -SF Gp120 -SF CA -SF RT -SF env Vpu -SF SP- Gp41 -SF 5′ LTR MA CA NC p6 pol vif nef NC-SF IN tat-SFvpu revNef -SF 3′ LTR pr rt int vpr gp120 gp41 p6-SF Tat -SF SF = 2x Strep 3x FLAG Rev -SF SP = Signal peptide Gag -SF Pol(-) -SF Vif -SF Gp160 -SF MA -SF PR(-)-SF Vpr -SF Gp120 -SF CA -SF RT -SF Vpu -SF SP- Gp41 -SF NC-SF IN -SF Nef -SF p6-SF Tat -SF SF = 2x Strep 3x FLAG Rev -SF SP = Signal peptide Supplementary Figure 1. Schematic representation of the HIV genome. The 3 polyproteins (Gag, Pol and Env (Gp160)), processed proteins (MA, CA, NC, p6, PR, RT, IN, Gp120 and Gp41), and accessory proteins (Vif, Vpr, Vpu, Nef, Tat and Rev) were cloned and affinity tagged (2xStrepTagII-3xFLAG (SF)) on their C-terminal ends. PR was mutated to be catalytically inactive (PR(-)), and Supplementary Figure 1. Schematic representation of the influenza HA signal peptide sequence was fused to the HIV genome. The 3 polyproteins (Gag, Pol and Env gp41. Sources and amino acid sequences of all factors, (Gp160)), processed proteins (MA, CA, NC, p6, PR, RT, many of which are codon-optimized, are in Supplemen- IN, Gp120 and Gp41), and accessory proteins (Vif, Vpr, tary Table 1 and Supplementary Methods. Vpu, Nef, Tat and Rev) were cloned and affinity tagged (2xStrepTagII-3xFLAG (SF)) on their C-terminal ends. PR was mutated to be catalytically inactive (PR(-)), and Supplementary Figure 1 the influenza HA signal peptide sequence was fused to gp41. Sources and amino acid sequences of all factors, many of which are codon-optimized, are in Supplemen- tary Table 1 and Supplementary Methods. Supplementary Figure 1 WWW.NATURE.COM/NATURE | 1 2 RESEARCH |WWW.NATURE.COM/NATURE ( proteinsafterbeingtransientlytransfectedintoHEK293cells analysis ofcelllysatesall18HIV-SF proteins. blotanalysisofall18HIV Western 2: Supplementary Figure a ) orinducedviatetracyclineinJurkatcellsstablyexpressing eachtaggedfactor( SUPPLEMENTARY INFORMATION SUPPLEMENTARY b a 100 – 150 – 250 – 100 – 150 – 250 – kDa kDa 15 – 20 – 37 – 50 – 75 – 15 – 20 – 37 – 50 – 75 – Gag GFP MA Gag CA MA NC CA p6 NC Pol p6 PR Pol RT PR IN RT Vif IN Vpr Vif Vpu Vpr Tat Vpu Rev Tat Nef Gp160 Rev Anti-FLAG westernblot Gp120 Nef Gp160 Gp41 Gp120 b ). Gp41 Supplementary Figure2 SUPPLEMENTARY INFORMATION RESEARCH IN Vif p6 RT a GFP Gag MA CA NC Pol PR Vpr Vpu Tat Rev Nef Gp160 Gp120 Gp41 kDa 200 – 150 – 120 – 100 – 85 – 70 – 60 – 50 – 40 – 30 – 25 – 20 – 15 – 10 – b RT IN Vif Vpr Vpu Tat Rev Nef Gp160 Gp120 Gp41 Gag MA CA NC p6 Pol PR kDa 200 – 150 – 120 – 100 – 85 – 70 – 60 – 50 – 40 – 30 – 25 – 20 – 15 – 10 – Supplementary Figure 3: Silver stained SDS-PAGE of HIV affinity purifications. Eluates of FLAG affinity purifications from both HEK293 (a) and Jurkat (b) cells were subjected to SDS-PAGE and stained with silver. The tagged HIV protein in each lane is marked by a black arrow. Supplementary Figure 3 WWW.NATURE.COM/NATURE | 3 RESEARCH SUPPLEMENTARY INFORMATION Average correlation coefficient correlation Average Supplementary Figure 4: Average correlation coefficients for replicate experiments for each HIV protein. For a given bait, we calculated cross-correlations between experiment vectors derived from all combinations of affinity tags and purification protocols, and then determined the average values (bar height) and standard deviations (error bars) of these correlations. The average correlation coefficient across all baits (0.68) is shown with black dashed line. Supplementary Figure 4 4 | WWW.NATURE.COM/NATURE elution-FLAG beads-FLAG elution-Strep beads-Strep gel-FLAG gel-Strep ducibility (node color) for each combination of affinity tagandisolationprotocol. ducibility (nodecolor)foreachcombinationofaffinity by theirabundances(nodesize)andrepro proteinpairsareshownclustered human-HIV All 39well-characterized pairs. protein and abundance of human-HIV the well-characterized 5: Reproducibility Supplementary Figure Vif-ELOC Vif-ELOB Vpr-DDB1 MA-SYHM Vpu-FBW1B gp41-GANAB gp120-CALX Vpu-SKP1 gp160-CALX Vpr-ANKH1 gp120-HLAA gp160-CALR Tat-AFF4 Tat-SIRT1 NC-APOBEC3F Tat-ENL gp160-CALR Tat-CCNT1 gp41-LMAN2 gp120-PDIA1 Vif-RBX2 Vpu-CUL1 Vif-CUL5 Vif-CUL2 Nef-ACOT8 Vpr-VPRBP gp160-LMAN1 gp120-GANAB gp160-GANAB gp160-HLAA gp41-LMAN1 INFORMATION SUPPLEMENTARY gp41-PDIA1 gp120-CALR gp41-CALR Nef-NMT1 WWW.NATURE.COM/NATURE | 5 | WWW.NATURE.COM/NATURE Vpr-DDA1 Tat-CDK9 Supplementary Figure 5 Vpu-FBW1A MA-SYK 0.002 Exp. reproducibility Exp. Abundance 0.02 7 6 5 4 3 2 1 RESEARCH 0.07 - RESEARCH SUPPLEMENTARY INFORMATION a HIV dataset - 39 known bait-prey pairs b HIV dataset - ribosomal proteins c MiST 0.75 cuto TPR FPR Supplementary Figure 6: Comparison of MiST, CompPASS and SAInt scoring systems to the HIV-human PPI dataset. Using the recall plots, MiST performs better than CompPASS and SAInt when counting (a) the number of 39 well-characterized human-HIV pairs (true positives) and (b) a set of 1596 interactions involving ribosomal proteins (false positives). The black lines correspond to the MiST score cut-off of 0.75. (c), Using the set of true and false positives (see above), an ROC assessment of the false-positive rates (FPR) and true-positive rates (TPR) reveals that MiST behaves superior when compared to COMPass and SAInt. The MiST score cut-off (0.75, marked) falls just before the plateau of the line. Supplementary Figure 6 6 | WWW.NATURE.COM/NATURE SUPPLEMENTARY INFORMATION RESEARCH a 0.60 b 28 0.55 26 0.50 24 0.45 22 0.40 20 0.35 Recall Interaction Known 18 Overlap with the Final Set Final the with Overlap 0.30 16 Isolation: gel beads elution all all elution beads all Isolation: gel beads elution all all elution beads all Tag: all all all strep flag flag flag all Tag: all all all strep flag flag flag all c 1.0 d 35 0.9 30 0.8 25 0.7 0.6 20 0.5 15 Known Interaction Recall Interaction Known Overlap with the Final Set Final the with Overlap 0.4 0.3 10 2 4 6 8 10 12 14 2 4 6 8 10 12 14 number of replicas number of replicas Supplementary Figure 7: Analysis of different experimental protocols and number of replicates. Sets of the bait-prey pairs obtained by resampling of different experimental protocols were compared to the final set of 387 HEK293-derived PPIs (a) and to the 39 well-characterized HIV-human PPIs (b). Similar analysis was carried out to study the overlap between sets obtained by sampling different number of replicas (c and d). Box plots identify the middle 50% of the data (box), the median (horizontal red line), median confidence interval (notch), and the extreme points (data points greater than 1•IQR shown with red circles). Supplementary Figure 7 WWW.NATURE.COM/NATURE | 7 RESEARCH SUPPLEMENTARY INFORMATION a DUB dataset b TIP49 dataset Supplementary Figure 8: Comparison of the MiST, CompPASS and SAInt scoring systems using the DUB (a) and TIP49 (b) datasets. Using two different datasets (DUB and TIP49) and two sets of well- characterized interactions, recall plots were generated to compare the three different scoring systems. The black lines correspond to the cut-offs used in the original studies36,37. 8 | WWW.NATURE.COM/NATURE Supplementary Figure 8 SUPPLEMENTARY INFORMATION RESEARCH 160 140 120 100 80 HEK293 only Density 60 Jurkat only 40 20 0 -4 -3 -2 -1 0 1 2 3 4 5 6 7 Gene expression difference (Jurkat - HEK293) Supplementary Figure 9: Distribution of the gene expression scores (measured by the difference in z scores of HEK293 and Jurkat cells) for each protein in HEK293 and Jurkat specific interactions. Note that proteins in HEK293 specific interactions (HEK293 only) tend to have shifted expression to the left indicative of higher expression in HEK293 cells, and similarly host proteins in Jurkat specific interactions (Jurkat only) have an expression distribution shifted towards right indicating a higher expression in Jurkat cells. The difference between the distributions was highly significant (p = 8.16 • 10-7; Mann-Whitney U test). WWW.NATURE.COM/NATURESupplementary Figure 9 | 9 RESEARCH SUPPLEMENTARY INFORMATION a GFP Gag MA CA NC p6 b GFP Gag MA CA NC p6 Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP TR150 OLA1 0.807 (HEK) 0.319 (Jurkat) 0.911 (HEK) 0.819 (Jurkat) SPCS SPCS 0.814 (HEK) 0.200 (Jurkat) 0.862 (HEK) 0.831 (Jurkat) NS1BP MCA3 0.753 (HEK) 0.260 (Jurkat) 0.907 (HEK) 0.764 (Jurkat) SF3B4 MCA1 0.748 (HEK) 0.123 (Jurkat) 0.919 (HEK) 0.740 (Jurkat) DDX49 MCA2 0.824 (HEK) 0.000 (Jurkat) 0.915 (HEK) 0.506 (Jurkat) ANM1 SDCB1 0.758 (HEK) 0.037 (Jurkat) 0.833 (HEK) 0.000 (Jurkat) OLA1 NOLC1 0.777 (HEK) 0.000 (Jurkat) 0.809 (HEK) 0.000 (Jurkat) E2AK2 SDCG8 0.766 (HEK) 0.000 (Jurkat) 0.781 (HEK) 0.000 (Jurkat) SPF45 0.765 (HEK) 0.000 (Jurkat) Supplementary Figure 10 page 1 of 2 10 | WWW.NATURE.COM/NATURE SUPPLEMENTARY INFORMATION RESEARCH c GFP Gag MA CA NC p6 d GFP Gag MA CA NC p6 Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP Input IP Gag ANM1 0.776 (HEK) GFP 0.164 (Jurkat) CA RINI FLAG MA 0.870 (HEK) NC 0.000 (Jurkat) p6 THUM1 0.822 (HEK) 0.000 (Jurkat) LRC47 0.814 (HEK) 0.000 (Jurkat) CSDE1 0.812 (HEK) 0.000 (Jurkat) STRAP 0.788 (HEK) 0.000 (Jurkat) MYPT1 0.785 (HEK) 0.000 (Jurkat) CTNA3 Supplementary Figure 10: Co-immunoprecipita- 0.781 (HEK) tions of interaction candidates using antibodies 0.000 (Jurkat) against the endogenous proteins.
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