Fig. 1. Src is a component of the TJ complex. (A) Western blots for ZO-1 and total Src after ZO-1 immunoprecipitation from QL12-MDCK cells in +/–dox conditions. ZO-1 hybridoma was added undiluted to an equivalent amount of cell lysates prepared as described in the Materials and Methods. Negative controls were rat ascites using IgG beads. Arrows indicate the ZO-1 and Src bands. (B) Lysates from G12- and QL12-MDCK cells (+/–dox) were immunoprecipitated with ZO-1 and probed with antibodies against active Src (pTyr419) and ZO-1. (C) Confocal microscopy of G12-MDCK cells +/–dox double stained for ZO-1 and Src as described in the Materials and Methods. (Ca,Ch) White box indicates the cell that was examined at higher magnification in the panels to the right. In x-y and lat-z panels, a region of the membrane was identified (white enclosure) for additional analysis. The lat-z images represent the apical 5 µm region of the cell (total height approximately 8 µm). (Ca-Cg) +dox; (Ch-Cn) –dox. FITC-ZO-1, Texas-red c-Src and merged images are shown for both conditions in x-y and lat-z orientation. Scale bars: 10 µm (Ca,Ch), 5 µm (Cb-Cg,Ci-Cn).