Interactions of cIAP1 with Signaling Proteins: Immunoprecipitation Analysis

1. Supplementary Figure 1 FLAG-Co FLAG-cIAP1 FLAG-L47A FLAG-Co FLAG-cIAP1 FLAG-L47A Input IP αFLAG 70 FLAG TRAF2 56 Figure S1. Immunoprecipitation analysis of the interaction of cIAP1 and the mutant L47A with TRAF2. 293T cells were transfected with indicated FLAG-constructs. FLAG was immunoprecipitated with specific antibody and the interaction was revealed using an anti-FLAG or an anti-TRAF antibody.

2. Supplementary Figure 2 siRNA: control RhoGDIα cIAP1: - + + - cdc42 21 23 70 70 RhoGDIα cIAP1 Figure S2. Immunoblot analysis of cdc42, RhoGDIα and cIAP1 in in NIH3T3 transfected with cIAP1 encoding vector plus Control or RhoGDIα-targeting siRNA. HSC70 is used as loading control. HSC70

3. 53 48 27 Supplementary Figure 3 A IP myc Input EGFP EGFP-Cdc42 EGFP-Rac1 EGFP-RhoA EGFP EGFP-Cdc42 EGFP-Rac1 EGFP-RhoA GFP B GST GST-cIAP1 GTPγS GTPγS GDP GDP lysate NT NT Figure S3. cIAP1 can interact with cdc42. (A) Co-immunoprecipitation analysis of the interaction of cIAP1 with RhoA, Rac1 and cdc42. HEK293T cells were transfected with myc-cIAP1 and EGFP, EGFP-RhoA, EGFP-Rac1 or EGFP-cdc42 encoding vector. Immunoprecipitation was performed using anti-myc antibody and interactions were revealed by immunobloting using anti-GFP antibody. (B) GFP-cdc42 from transfected HEK293T cell lysates was charged with GDP or GTP, and incubated with GST or GST-cIAP1 immobilized onto glutathione sepharose beads. Interactions were revealed by immunoblotting using specific GFP antibody. 48 GFP-Cdc42 GST-cIAP1 95 26

4. Supplementary Figure 4 * * Filopodia (fold induction/Co) 70 72 70 siRNA: Co cIAP1 cIAP2 cIAP1 cIAP2 HSC 70 siRNA: Co cIAP1 cIAP2 Figure S4. Silencing of cIAPs inhibits TNFα-mediated Filopodia formation. NIH3T3 fibroblasts were transfected with control (Co), cIAP1 or cIAP2 targeting siRNA, serum starved for 16 hours and stimulated for 15 min. with 100ng/mL TNFα. Left panel: The efficiency of siRNA was checked by immunoblot analysis. HSC70 is used as loading control. Right panel: Filopodia were quantified by counting cells exposing more than 5 filopodia. > 100 cells were analyzed. Results were expressed as fold filopodia induction/untreated cells transfected with control siRNA. Mean ± sd of three independent experiments. Statistical analysis performed using the Student’s t-test. *: p<0,05.

5. Supplementary Figure 5 wt cIAP1-/- NT • 125 • 100 • 75 • 50 • 25 • 00 Survival (% ) TNF 4h TNF 8h Figure S5. MEF cIAP1-/- are sensitive to TNF-induced cell death. MEF wt or cIAP1-/-were serum starved for 16 hours, then stimulated for 4 or 8 hrs with 100ng/mL TNF. Cell viability was evaluated by a crystal violet staining (Left panel: One representative experiment is shown) and quantified after elution by a colorimetric analysis (Right panel: Mean ± sd of three independent experiments).

6. Supplementary Figure 6 GFP-Co GFP-L47A GFP-cIAP1 GFP-Co GFP-L47A GFP-cIAP1 Input IP αGFP cIAP1 GFP 96 70 GFP-cIAP1/L47A Endogenous cIAP1 96 70 Lower exposure GFP-cIAP1/L47A Heavy chains GFP 96 26 Figure S6. HEK293T cells were transfected with GFP, GFP-cIAP1 or GFP-cIAP1L47A (GFP-L47A). GFP-IAP constructs were Immunoprecipitated using anti-GFP antibody and interactions were revealed by immunobloting using anti-cIAP1 antibody detecting the overexpressed constructs and endogenous cIAP1.

7. Supplementary Figure 7 *** ** ** *** Filopodia (fold inductin / UT) si-Co si-TRAF2 Co Co cIAP1 MEFs: wt cIAP1-/- wt Figure S7. MEFs from wt or cIAP1-/- mice were transfected or silenced as in Figure 5C-E, serum starved, and treated for 10 minutes with 100ng/mL EGF. Filopodia were evaluated by a Microscopic analysis of F-actin immunostained using AlexaFluor488-conjugated Phalloidin and counted. > 100 cells were analyzed. Mean ± sd of three independent experiments. Statistical analysis performed using the Student’s t-test. ***: p<0,001; **: p<0,01; *: p<0,05.

8. Supplementary Figure 8 Figure S8. Nude mice were injected in the tail vein wih 1.106 MEF wt or cIAP1-/- expressing Hras-V12 and the mice were sacrificed 2 weeks later. Pictures of the whole lungs (upper panel) or lung sections stained with hematoxylin and eosin (lower panel) are presented.

9. Supplementary Figure 9 Figure S9. CFSE-labeled wt MEFs expressing HRas-V12 were added onto confluent HUVECs and filmed for 300 min. by time-lapse microscopy (picture every 15 min.) The intercalated cells that display a non-round shape and a low phase-bright were marked with an Asterisk. The arrow indicates the cell that did not interacalate

10. Supplementary Figure 10 cIAP1: - - - + - - + His-Ub: + - + + - + + HA-cdc42: - wt wt wt L61 L61 L61 130 Co2+ Pull down HA (ubiquitin) 72 43 26 cIAP1 72 HA 26 Figure S10. HEK293T were transfected with His-ubiquitin, HA-cdc42, HA-cdc42L61 (L61) constitutive active mutant of cdc42 and cIAP1. His-ubiquitin was precipitated by using cobalt beads and ubiquitinated proteins were revealed by immunobloting by using anti-HA specific antibody. The efficiency of transfection was checked by immunobloting by using anti-cIAP1 and anti-HA antibodies.