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Fig. 6. Endogenous G ${alpha}$ 12 is activated during TJ assembly in the calcium switch and thrombin delays TJ assembly. (A) Calcium switch in control G ${alpha}$ 12-MDCK cells +dox. Cells were cultured overnight in low calcium (LC; t=0) medium and then switched back to normal calcium (NC) medium. TER measurements were obtained at the specified intervals. Thrombin (2 U/ml) was added at t=0. Results are the mean ± s.e.m. for n=4 at each time point. The experiment was repeated three times with nearly identical results. (B) Western blot of G ${alpha}$ 12 +dox cells +/–thrombin analyzed by GST-TPR pull-down at the specified times after the calcium switch. (C) Calcium switch in G ${alpha}$ 12-MDCK cells with induced G ${alpha}$ 12 expression (–dox). (D) Western blot of G ${alpha}$ 12 –dox cells +/–thrombin analyzed by GST-TPR pull-down at the specified times after the calcium switch. The first lane of the top panel shows the induced G ${alpha}$ 12 and is $~$ 10% of the protein used for the pull-downs. (E) Western blots for G ${alpha}$ 13 on G ${alpha}$ 12-MDCK cells +/–dox and +/–thrombin and pulled-down with GST-TPR as described in C. For each panel, the +/–thrombin results were obtained from a single gel.

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