Fig. 6. Characterization of the effects of p95-C3 and p95-K39 expression in fibroblasts. (A) Cells transfected with pFlag-p95-C3 were stained for p95-C3 (a,c) and F-actin (b,d). The same cells are shown in a,b and in c,d, respectively. Cells cotransfected with pFlag-p95-C3 and pCMV6m/Pak1 (e,f), and with pFlag-p95-C3 and pXJ40-HA-ßPIX (g) were immunostained to detect p95-C3 (e), PAK1 (f) and ß-PIX (g). Colocalization of p95-C3 (h) with Rab11 (i). In (j), the distribution of p95-C3 (green) and that of EEA1 (red) are compared. Bars, 10 µm. (B) Identification of the ß-PIX/p95-K39/paxillin complex. Fibroblasts cotransfected with pFlag-p95-K39 and pXJ40-HA-ßPIX were lysed and incubated first with control Protein-A-Sepharose beads (left lane), and then with beads coated with the anti-Flag mAb (right lane). After SDS-PAGE and blotting, filters were incubated with anti-Flag mAb (top), anti-PIX pAb (middle), and anti-paxillin mAb (bottom). Molecular weight markers are indicated to the left of the blot. (C) Cells transfected with pFlag-p95-K39 were analyzed for the distribution of p95-K39 (a), and F-actin (b); c shows the localization ß-PIX at large vesicles in a cell cotransfected with pFlag-p95-K39 and pXJ40-HA-ßPIX; d shows the localization at large vesicles of PAK in a cell cotransfected with pFlag-p95-K39 and pCMV6m/Pak1. Bars, 10 µm.
