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Fig. 8. Rac1 activity is enhanced by ZRP-1 depletion. (A) Rac1 pull-down assay. Lysates from HeLa cells treated with or without ZRP-1 siRNA were immunoblotted with anti-ZRP-1 antibody (lower panel) and anti-Rac1 antibody (middle panel). The upper panel shows GTP-bound Myc-Rac1 precipitated with GST-PAK CRIB. (B) Effects of expression of the dominant-negative mutant of Rac1. HeLa cells were transfected with or without Cy3-labeled ZRP-1-siRNA. Twenty-four hours later, these HeLa cells were transfected with expression vectors for Myc-DN-Rac1 and EGFP-actin at a ratio of 9:1. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals. Bars, 15 µm. (C) Exogenously expressed Flag-tagged-p1130Cas from control or ZRP-1-depleted HeLa cells was analyzed with a phosphotyrosine antibody (4G10). (D) Endogenous Crk II was immunoprecipitated and analyzed as in (C). (E) The relative levels of total Tyr phosphorylation of Flag-p130Cas and Crk II in ZRP-1-depleted cells as compared with those in control cells. (F) Effects of overexpression of wild-type RhoA. HeLa cells were transfected with or without Cy3-labeled ZRP-1-siRNA. Twenty-four hours later, these HeLa cells were transfected with expression vectors for Myc-WT-RhoA and EGFP-actin at a ratio of 9:1. Beginning 48 hours after vector transfection, cells were imaged at 1-minute intervals. Bars, 15 µm.
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