JCS028647 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Supplementary Fig. S1. Effect of Vav2-, Tiam1- and Asef-knockdown on EGF-induced activation of Rac1 and Cdc42. (A, B) A431 cells transfected with siRNA targeting Vav2, Tiam1 and Asef or with control siRNA were starved for 6-12 hours, not treated or treated with 25 ng/ml EGF for 2 minutes, and examined by pull-down assay for active (A) Rac1 or (B) Cdc42. Representative images are presented from each experiment performed at least three times. The summary of these data is given in Figs 1D, 2G and 3B in the main text.

• Supplemental Figure S2 (Adobe PDF) -

  Supplementary Fig. S2. EGF-induced phosphorylation of EGFR and Vav2 in A431 cells. A431 cells were starved for 12 hours and then left unstimulated or stimulated with 25 ng/ml EGF for 5 minutes. (A) Cell lysates were immunoblotted using antibodies against phosphotyrosine (anti-PY20), EGFR (anti-EGFR), phospho-Vav2 (anti-pY172-Vav) and Vav2 (anti-Vav2). Representative images are shown from experiments performed at least three times. (B) Phosphorylated Vav2 versus unphosphorylated Vav2 with and without EGF stimulation is expressed the average fold increase ± s.d. *P<0.05, significantly difference as compared with mock-treated cells (student’s t-test).

• Supplemental Figure S3 (Adobe PDF) -

  Supplementary Fig. S3. Effect of Sos knockdown on EGF-induced Ras activation. A431 cells were transfected with an empty pSuper vector (control) or pSuper-Sos1 and pSuper-Sos2 (Sos1, 2 KD). After puromycin selection, cells were further transfected with pRaichu-Ras. After 3-6 hours of serum starvation, cells were stimulated with 25 ng/ml EGF. The effect of the Sos knockdown on Ras activation was determined as in Fig. 1. Error bars indicate ± s.d (n≥3).

• Supplemental Figure S4 (Adobe PDF) -

  Supplementary Fig. S4. Effect of Asef knockdown on the EGF-induced Rac1 and Cdc42 activation in HeLa cells. HeLa cells were transfected with an empty pSuper vector (control) or pSuper-Asef. After puromycin selection, cells were further transfected with pRaichu-Rac1 (left graph) or pRaichu-Cdc42 (right graph). After 6 hours of serum starvation, cells were stimulated with 25 ng/ml EGF. From the peak values of the normalized ratio, the effect of Asef knockdown was calculated as described in the legend to Fig. 1D. The cell numbers examined for each condition are as follows: (left graph) control, n=15; Asef1 KD, n=11; (right graph) control, n=11; Asef1 KD, n=20. Error bars indicate ± s.d. *P<0.05, significant difference compared with control cells (student’s t-test).

• Supplemental Figure S5 (Adobe PDF) -

  Supplementary Fig. S5. Inhibition of EGF-induced activation of Rac1 and Cdc42 using dominant-negative mutants. (A,B) A431 cells were transfected with expression vectors of Raichu-Rac1 (control) and Cdc42-N17 mutant (Cdc42DN) and imaged and analyse as described in Fig. 2. (C,D) Experiments were performed as as described for panels A and B, except that A431 cells were transfected with expression vectors of Raichu-Cdc42 (control) and the Rac1-N17 mutant (Rac1DN). Error bars indicate the ± s.d (n≥3)

• Supplemental Figure S6 (Adobe PDF) -

  Supplementary Fig. S6. Effect of phosphorylation of Asef on its binding to APC. A431 cells were transfected with plasmids expressing Myc-tagged APC together with HA-tagged wild-type Asef or the Asef mutants carrying amino acid mutations Tyr94 or Tyr104 to either Phe or Glu (Y94F, Y94E or Y104F, Y104E, respectively). Asef proteins were immunoprecipitated using anti-HA antibody. The immunoprecipitates and total cell lysates were analyzed by SDS-PAGE and immunoblotting was carried out using anti-Myc and anti-HA antibodies.