Fig. 5. Pharmacological inhibition of PKC activity impairs stress fibre and focal adhesion formation but not cell spreading of REFs on FN. (A) REF cells growing under standard conditions were incubated with the indicated amounts of PKC inhibitors for 30 minutes and lysed for western blotting. Equal amounts of lysates were resolved by SDS-PAGE and probed with antibodies against pT-250, pT-638, pS-657 and total PKC as a loading control, as indicated. (B) Serum-starved REFs were trypsinized and treated with 0.5 µM Gö6976 in suspension for 20 minutes before plating on FN. At 2.5 hours cells were fixed, permeabilized and stained for F-actin and vinculin. (C) REFs were incubated with cell-permeable C3 transferase (TAT-C3) protein (45 µg/ml) in growth medium overnight at 37°C. Trypsinized REFs were replated on FN for 2.5 hours, fixed with 4% paraformaldehyde, permeabilized and stained for F-actin and paxillin. Bar, 50 µm.