Fig. 7. Loss of PKC results in impaired Rac signaling in fibroblasts. (A) Rac is expressed equally in PKC^+/+ (WT) and PKC^-/- fibroblasts. Cells were loaded with or without GTPS (positive control, to assess maximal Rac activation) and in the presence or absence of TGFβ (4 ng/ml, 1 hour). Cell lysates were subjected to SDS/PAGE and western blot analysis with an anti-Rac antibody. Experiments were performed six independent times. Quantitiative densitometry data are indicated on the right. (B) Rac activity assay. Input protein assessed in parallel with A was used in the standard Rac activity assay, as described in the Materials and Methods. Rac-GTP was immunoprecipitated from cell lysates. The precipitated Rac-GTP was detected by immunoblot analysis using anti-Rac. GTPS (lane 1), GDP (lane 2), PKC^+/+ (WT, lane 3), PKC^+/+ (WT)+ TGFβ (lane 4); PKC^-/- (lane 5); PKC^-/- + TGFβ (lane 6). Experiments were performed three independent times. Quantitative densitometry data are indicated on the right (^*P<0.05 relative to wild-type control).