Fig. 9. Thrombin stimulates the loss of barrier function in intact monolayers through G12, Src and Hsp90. (A) Confluent monolayers of G12-MDCK cells in +dox on Transwell filters were stimulated with thrombin (2 U/ml) at t=0 and TER was measured over 12 hours. Results are the mean ± s.e.m. of n=4 and were normalized to the untreated control. (B) Monolayers were stimulated at t=0 with thrombin (2 U/ml), PP2 (10 µM), GA (0.2 µM), thrombin plus PP2 or GA, bradykinin (100 µM), or bradykinin plus PP2 or GA and compared to vehicle (control). TER was measured at numerous time points. The results (normalized to the untreated control) at 40 minutes are shown (n=4 ± s.e.m.). ^*P<0.05. (C) Confluent G12-MDCK cells in +dox were stimulated at t=0 with thrombin and lysates were prepared at t=0 (no thrombin), 15 and 30 minutes of stimulation for analysis by GST-TPR pull-down. Western blots to G12 and Hsp90 were performed as described above.