The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein  Ben N.G. Giepmans, Wouter H. Moolenaar  Current Biology  Volume 8, Issue 16, Pages 931-934 (July 1998) DOI: 10.1016/S0960-9822(07)00375-2

Figure 1 Schematic representation of (a) Cx43 and (b) ZO-1. Consensus protein domains and regions with putative signalling functions are indicated. Abbreviations in Cx43: TM1–4, transmembrane domains 1–4; Y265, Src phosphorylation site; PP, proline-rich sequence that binds the SH3 domain of v-Src [4]; DDLEI, carboxy-terminal sequence involved in ZO-1 binding. For ZO-1, the three PDZ domains and one SH3 domain are indicated. Abbreviations in ZO-1: GUK, guanylate kinase homology domain; α, alternative splicing region; PP, proline-rich domain. Note that the prey sequence contains the entire PDZ2 domain and flanking sequences. Current Biology 1998 8, 931-934DOI: (10.1016/S0960-9822(07)00375-2)

Figure 2 Association of Cx43 with ZO-1 depends on the integrity of the Cx43 carboxyl terminus. COS7 cells were transfected with empty vector or with the indicated Cx43 cDNAs encoding wild-type Cx43 (WT), carboxy-terminally Myc-tagged Cx43 (tag) and Cx43 lacking the carboxy-terminal isoleucine residue (Δ). The total lysate and the immunoprecipitates (IP) of endogenous ZO-1 from these cells were analysed by western blotting using anti-ZO-1 or anti-Cx43 antibodies. Molecular weights are in kDa. (a) Wild-type Cx43 coimmunoprecipitated with endogenous ZO-1 (compare lane 2 with the empty vector control in lane 1). When the carboxyl terminus was modified by adding a Myc epitope tag, the association was abolished (compare lane 3 with lane 2). (b) Wild-type Cx43 coprecipitated with ZO-1, whereas the Cx43 deletion mutant that lacks the carboxy-terminal isoleucine failed to coprecipitate (compare lane 2 with lane 3). The position of the contaminating immuno-globulin G (IgG) from the immunoprecipitation procedure is indicated to the right of the blots. Current Biology 1998 8, 931-934DOI: (10.1016/S0960-9822(07)00375-2)

Figure 3 Association between endogenous Cx43 and ZO-1 in (a) Rat-1 fibroblasts and (b) Mv1Lu mink lung epithelial cells. (a) ZO-1 and Cx43 were immunoprecipitated from Rat-1 cells, and subjected to western blotting using antibodies for ZO-1 (upper panel) and Cx43 (lower panel). Control samples (C) that were precleared with normal rabbit serum and normal mouse serum are shown in lanes 2 and 4, respectively. A shorter exposure of the Cx43 immunoprecipitation (IP) is shown compared with the ZO-1 immunoprecipitation. Both non-phosphorylated and phosphorylated forms of Cx43 are detected in the ZO-1 immunoprecipitate (lane 3); the phosphorylated form migrates more slowly [3–5]. (b) ZO-1 was immunoprecipitated from mink lung epithelial cells. The upper panel shows the presence of ZO-1 in the anti-ZO-1 immunoprecipitate. The lower panel shows the coimmunoprecipitation of Cx43 with ZO-1 (lane 2). Lane 3 represents a control sample (C) precleared with rabbit anti-mouse immunoglobulin. Current Biology 1998 8, 931-934DOI: (10.1016/S0960-9822(07)00375-2)

Figure 4 Colocalisation of endogenous Cx43 (green) and ZO-1 (red) in Rat-1 cells, as represented by the yellow regions in the merged image. Results from two different analyses are shown, with the lower panels representing only part of a cell as visualised using higher magnification to reveal punctate staining. Note that ZO-1 is localised to gap-junctional plaques (typical punctate staining) and is also found at other regions of cell–cell contact where Cx43 is not detected. Note the location of both ZO-1 and Cx43 at regions of cell–cell contact (more evident in the upper panels). Current Biology 1998 8, 931-934DOI: (10.1016/S0960-9822(07)00375-2)