Fig. 6. Endogenous G12 is activated during TJ assembly in the calcium switch and thrombin delays TJ assembly. (A) Calcium switch in control G12-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 G12 +dox cells +/–thrombin analyzed by GST-TPR pull-down at the specified times after the calcium switch. (C) Calcium switch in G12-MDCK cells with induced G12 expression (–dox). (D) Western blot of G12 –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 G12 and is 10% of the protein used for the pull-downs. (E) Western blots for G13 on G12-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.