Fig. 5. Hsp90 is a component of the activated G12 complex and is necessary for Src activation and ZO-2 phosphorylation. (A) Src activation in QL12-MDCK cells +/–dox and +/–GA. Identical amounts of cell lysate were analyzed by western blotting with antibodies specific for pTyr-419 as described in the Materials and Methods. Membranes were stripped and reanalyzed for total Src and occludin. (B) Band intensity was quantified as described above and the pTyr-419 immunoreactivity normalized to total Src. The relative amount of pTyr419 for –dox and –dox +GA is shown in comparison to the +dox condition. (C) Phosphotyrosine content of immunoprecipitated ZO-2 from QL12-MDCK cells +/–dox and +/–GA. ZO-2 was immunoprecipitated and analyzed with 4G10 anti-phosphotyrosine as described above. (D) Interaction of G12 with ZO-1 in QL12-MDCK cells +/–dox and +/–GA. ZO-1 was immunoprecipitated under the specified conditions and precipitates were analyzed for G12 by western blotting. Control is lysate with beads alone. (E) Steady-state protein levels of TJ proteins and G12 in QL12-MDCK +/–dox and +/–GA (2 µM). (F) Validation of GST-TPR pull-down of activated G12. QL12- and G12-MDCK cells were cultured in –dox, and lysates (800 µg) were pulled-down with 1 µg GST or GST-TPR, as described in the Materials and Methods, and analyzed by western blotting for G12. 10% of the input of wild-type G12 is shown in the lysate lane. (G) Hsp90 western blot of GST-TPR pull-downs from G12- and QL12-MDCK cells in –dox.