(A) Schematic, linear diagrams of Cx36 and ZO1 homologues. Domains are depicted as gray shapes; TM = transmembrane, PDZ, SH3, GUK, and ZU5 = protein-protein interaction modules; hs = Homo sapiens, dr = Danio rerio. Amino acid alignments are shown for the indicated expanded regions. Black bars represent conserved amino acids; non-conserved amino acids are indicated. Maroon boxed amino acids represent the conserved PDZ-binding motif (PBM) of Cx36-family proteins (top) or the predicted PDZ1 residues of the conserved ligand-binding cleft of ZO1-family proteins (bottom). (B) HEK293T/17 cells were transfected with plasmids to express mVenus-ZO1b and either full-length Cx34.1 (lane 1), Cx34.1-∆PBM (lane 2), full-length Cx35.5 (lane 3), or Cx35.5-∆PBM (lane 4). 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. (C) Bacterially purified GST (lane 1), GST-Cx34.1-tail (lane 2), GST-Cx34.1-tail-∆PBM (lane 3), GST-Cx35.5-tail (lane 4), or GST-Cx35.5-tail-∆PBM (lane 5) was immobilized on glutathione beads and incubated with purified ZO1b PDZ1 domain. The tail regions used are depicted in the expanded regions in (A). Bound proteins were analyzed by immunoblot for the presence of ZO1b PDZ1 using anti-TEV cleavage site antibody (top, magenta). Equal loading of GST proteins is indicated by Coomassie staining (bottom, 2% input). Results are representative of three independent experiments. (D) Zebrafish brain extract from wt (lanes 1,2) or tjp1b/ZO1b-/- mutant (lanes 3,4) animals was immunoprecipitated with control whole mouse IgG (lanes 1,3) or anti-ZO1 antibody (lanes 2,4). Immunoprecipitates were analyzed by immunoblot for the presence of ZO1 using anti-ZO1 antibody (top, magenta), Cx34.1 using Cx34.1-specific antibody (middle, yellow), and Cx35.5 using Cx35.5-specific antibody (bottom, cyan). Asterisks (*) indicate antibody light chain. Results are representative of three independent experiments.
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