Supplementary Figure 1 IP: IgG HA 3HA-E2F1: cIAP1: wt K185 170

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Supplementary Figure 1 IP: IgG HA 3HA-E2F1: cIAP1: wt K185 + - + + - 170 IB : Ub (FK2) IB: HA IB: cIAP1 70 70 (3HA-E2F1) 70 Input cIAP1 HSC70 70 70 Supplementary Figure 1: Ubiquitination profile of E2F1 in HeLa cells co-transfected with pCMV-3HA-E2F1 wt or K185R (C) mutant, and His-tagged ubiquitin in the presence or absence of cIAP1 encoding vector. E2F1 was immunoprecipitated using anti-HA antibody and ubiquitin revealed using anti-poly-ubiquitin chain antibody (FK2). The expression of the transgenes was checked by a western blot analysis. β-actin was used as loading control.

Supplementary Figure 2 IP: E2F1 Ig 3HA-E2F1 : - K/R wt wt - wt HA-DP1 : - + + - + + IB: HA 70 (3HA-E2F1) 53 (HA-DP1) Input IB: HA E2F1 70 (3HA-E2F1) 53 (HA-DP1) 70 (3HA-E2F1) Supplementary Figure 2: Immunoprecipitation analysis of the interaction of DP1 with E2F1 wt or K161/164R mutant (K/R). Hela cells were transfected with 3HA-E2F1 encoding constructs and HA-DP1 encoding vector. E2F1 were immunoprecipitated using an anti-E2F1 antibody and DP1-E2F1 interaction was revealed by a western blot analysis by using anti-HA antibody.

Supplementary Figure 3 A B. HeLa C. U2OS D. HeLa IP: IgG E2F1 IB : Ub (FK2) IB: E2F1 V V Rb Input Rb α-tubulin B. HeLa 170 70 70 (3HA-E2F1) 110 55 G1 (%): S (%): G2 (%): S median (AU): si-Cyclin A 11.6 61.7 23.9 104 649 Si-Rb 32.6 38.0 38.6 97 324 si-Co 20.6 59.0 20.0 85 811 DNA content Cell count (AU) C. U2OS IB : Ub (FK2) IB: E2F1 si-RNA: Co Co cyA Rb Input Rb Cyclin A α-tubulin IP: IgG E2F1 55 110 170 70 70 (3HA-E2F1) GST GST E2F1 Rb cIAP1 Input - + - + + + + + + - + - - Pull-down D. HeLa 110 70 86 26 Supplementary Figure 3: A. Cell cycle analysis of U2OS cells 48 hours after transfection with co, cyclin A or Rb siRNA. The percentage of cells in cell cycle phase and the median of cell in S phase are indicated (upper) B-C. Influence of Rb on E2F1 ubiquitination. HeLa cells were co-transfected with Rb-encoding vector (B) or U2OS cells with cyclin A or Rb siRNA (C), and with pCMV-3HA-E2F1 and His-tagged ubiquitin wt. E2F1 was immunoprecipitated using anti-E2F1 antibody and ubiquitin revealed using anti-poly-ubiquitin chain antibody (FK2). The expression of the transgenes and the efficiency of siRNA were checked by a western blot analysis (lower panels). β-actin or α-tubulin was used as loading control. D. GST-pull down analysis of the interaction of GST-E2F1 with Rb in the presence of increasing quantity of cIAP1.