Figure 11.

Immunoblots demonstrating co-IP of Cx45/ZO-1 from retina and detection of Cx45 with polyclonal anti-Cx45. A, Cx45 detection by monoclonal anti-Cx45 in lysates of Cx45-transfected HeLa cells (lane 2) and in homogenate of mouse retina (lanes 4 and 7), but not in empty vector-transfected HeLa cells (lane 1) or in liver (lane 3). After IP of ZO-1 from retina homogenate using polyclonal anti-ZO-1, monoclonal anti-Cx45 detects Cx45 (lane 6) comigrating with Cx45 from retina homogentate (lane 4) and from Cx45-transfected HeLa cells (lane 2), but not in IP material after omission of anti-ZO-1 during the IP procedure (lane 5) or after IP of ZO-1 from retina using anti-FLAG as control IgG (lane 8). B, C, Polyclonal anti-Cx45 detection of Cx45 as a single band using 12.5% SDS-gel (B, lane 1) and as a doublet of bands using 10% SDS-gel (C, lane 1) in lysates of Cx45-transfected HeLa cells, and absence of these bands in empty vector-transfected HeLa cells (B, C, lane 2). D, IP of Cx45 from retina with monoclonal anti-Cx45, showing detection of a Cx45 band with polyclonal anti-Cx45 in IP material (lane 3), corresponding to a Cx45 band detected by the polyclonal antibody in lysate from Cx45-transfected HeLa cells (lane 2), and absence of the band in empty vector-transfected HeLa controls (lane 1) and after omission of monoclonal anti-Cx45 during the IP procedure (lane 4).