Figure 7. RhoA regulates the activation of Pak and its interaction with paxillin at the adhesome complex during contractile stimulation .

A, pak Thr423 phosphorylation was measured in tissues expressing RhoA T19N or sham‐treated tissues with or without ACh stimulation (n = 6). B, paxillin Tyr118 (Y118) phosphorylation and paxillin Ser273 (S273) were measured in tracheal smooth muscle tissues expressing the RhoA T19N mutant and in sham‐treated tissues after 5 min ACh stimulation or in unstimulated tissues (US) (n = 3). C, in situ PLA was used to analyse the interaction of paxillin and Pak1 in freshly dissociated differentiated canine tracheal smooth muscle cells. PLA fluorescence and phase contrast images are shown for each unstimulated (US) and ACh‐stimulated cell. In cells from sham‐treated tissues, the mean number of PLA spots was significantly higher in ACh‐stimulated cells than in unstimulated cells (n = 20 for ACh, n = 16 for US). In cells from RhoA T19N‐treated tissues, the ACh‐induced increase in the mean number of PLA spots was very small and was significantly inhibited (n = 21 cells for ACh, n = 15 cells for US). Cells were dissociated from tissues obtained from three separate experiments. D, freshly dissociated tracheal smooth muscle cells were double stained for paxillin and Pak1 to determine the effect of ACh stimulation on the localization of Pak1 and paxillin. US, unstimulated cells. A representative cell from each group of eight cells is shown. Values are means ± SEM. ^*Significant difference between ACh‐stimulated tissues, P < 0.05.