Fig. 7. PKC mediates signaling from integrin 5 to focal adhesion molecules. (A) Cells were trypsinized, collected, suspended into DMEM-H-1% BSA, and rotated (60 rpm) at 37°C for 45 minutes. Then half of cells was kept in suspension and the other half was replated onto fibronectin-coated (10 µg/ml) 60 mm dishes for 2 hours. After incubation, whole cell lysates were prepared for immunoblots for the indicated molecules. (B) Whole cell extracts from the cells without or with OXO treatment were prepared and immunoprecipitated with anti-integrin 5 (clone P1D6). The immunoprecipitates and lysates were immunoblotted for pS⁶⁴³PKC or PKC. (C) Whole cell extracts from RIE1-5 cells were immunoprecipitated with anti-PKC. The PKC immunoprecipitates were incubated in a reaction buffer (25 mM Tris, pH 7.5, 10 mM MgCl₂, 50 µM ATP and 1 mM DTT) without or with the PKC-depleted extracts (Extracts^*; 10 µg protein/reaction) in the absence or presence of 10 µM OXO for 30 minutes at 25°C with shaking. After incubation, SDS-PAGE sample buffer was added to stop the reaction before immunoblotting for pS⁶⁴³PKC and PKC (upper panels). For in vitro PKC kinase assay (lower panel), determination of phosphorylation of Ser/Thr in the substrate by recombinant PKC (recPKC) using HTScan^TM PKC kinase assay kit was performed following the manufacturer's protocols. The primary antibody of the kit and proper secondary antibody were used for immunoblots. (D) RIE1-5 cells were infected with adenovirus (Ad-Virus) encoding for a control protein (i.e. GFP, Cont) or WT PKC for 12 hours. Twenty four hours after the infection, cells were maintained in suspension (Sus) or replated onto fibronectin-coated (10 µg/ml; Fn) 60 mm dishes for 2 hours prior to cell lysates preparation and standard western blotting for the indicated molecules. (E,F) Whole cell lysates prepared as in Fig. 4C,D were normalized and used for standard western blotting for the indicated molecules. (G) RIE-4 or parental RIE1 WT cells in normal culture were harvested for integrin 4 and -tubulin immunoblots (upper panel). RIE1-4 cells were infected with control (Ad-cont) or DN PKC (Ad-DN PKC) adenovirus and treated with OXO (10 µM for 24 hours) prior to harvesting and immunoblotting for the indicated molecules. Data shown are representative of at least three independent experiments.