Figure 6-figure supplement 1

Figure 6-figure supplement 1

(A) HEK293T/17 cells were transfected with plasmids to express full-length Cx34.1 (lanes 1,2), Cx34.1-∆PBM (lanes 3,4), full-length Cx35.5 (lanes 5,6), or Cx35.5-∆PBM (lanes 7,8) together with empty vector (-) or mVenus-ZO1b (+). Lysates were immunoprecipitated with anti-GFP antibody and analyzed by immunoblot for the presence of mVenus-ZO1b using anti-GFP antibody (upper, magenta), Cx34.1 protein using Cx34.1-specific antibody (middle, yellow), or Cx35.5 protein using Cx35.5 specific antibody (middle, cyan). Total extracts (bottom, 5% input) were blotted for Connexin proteins to demonstrate equivalent expression and uniform antibody recognition of expressed proteins. Results are representative of three independent experiments. (B) Direct interaction of Connexin PBMs and the ZO1b PDZ1 domain by overlay assay. Bacterially purified GST (lanes 1,6), GST-Cx34.1-tail (lanes 2,7), GST-Cx34.1-tail-∆PBM (lanes 3,8), GST-Cx35.5-tail (lanes 4,9), GST-Cx35.5-tail-∆PBM (lanes 5,10), GST-Cx43-tail (lane 11), and GST-Cx43-tail-∆PBM were resolved by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were incubated with 1 µM ZO1b PDZ1 domain (top left), 1 µM ZO1b PDZ2 domain (top right) or buffer alone (middle, mock overlay). Bound proteins were analyzed by immunoblot for the presence of ZO1b PDZ using anti-TEV cleavage site antibody (top and middle). Equal loading of GST proteins is indicated by Stain-Free technology (bottom). Results are representative of three independent experiments. (C) Zebrafish brain extract from wt (lane 1), tjp1b/ZO1b^-/- (lane 2), or tjp1a/ZO1a^-/- (lane 3) animals were immunoprecipitated with anti-ZO1 antibody. Immunoprecipitates were analyzed by immunoblot for the presence of ZO1 using anti-ZO1 antibody (top, magenta) or Cx34.1 using Cx34.1-specific antibody (bottom, yellow). Asterisk (*) indicates antibody light chain. Results are from one independent experiment.