Colocalization and interactions of Vangl2 with BTB-associated proteins in the testis or Sertoli cells cultured in vitro with an established functional TJ-permeability barrier. A, Colocalization of Vangl2 (green fluorescence) with F-actin (red fluorescence) and the actin-regulatory proteins Eps8 (red fluorescence, an actin barbed end capping and bundling protein) and Arp3 (red fluorescence, a branched actin polymerization protein by converting a linear actin microfilament to a branched configuration via barded end nucleation) at the apical ES in the seminiferous epithelium of adult rat testes using paraffin sections fixed in paraformaldehyde. Scale bar, 20 μm, which applies to other micrographs. B, Colocalization of Vangl2 (green) with F-actin (red), TJ integral membrane protein claudin 11 (red), TJ adaptor protein ZO-1 (red), and polarity protein Scribble (red) at the basal ES/BTB in adult rat testes. Nuclei were visualized with DAPI (blue fluorescence). The relative location of basement membrane was annotated by a dashed white line, and the BTB was annotated by yellow arrowheads. Scale bar, 40 μm, which applies to other micrographs. C, Co-IP using testis lysates (∼1 mg of protein per lane) was performed using a goat anti-Vangl2 antibody (see Table 1) vs normal goat IgG (control), and the binding partner proteins in the pulled-out immunocomplexes were identified using corresponding specific antibodies against actin, actin regulatory proteins Eps8 and Arp3, and other BTB-associated proteins Scribble and N-cadherin (animal species used to generate the antibody were annotated). Vangl2 was found to interact with Scribble, but not actin, Eps8, Arp3, and N-cadherin (left panel), using testis lysates with normal goal IgG served as the negative control, and testis lysate at 40 μg protein without Co-IP served as the corresponding positive control. Specific interaction of Vangl2 with Scribble was confirmed by pulling out Vangl2 using an anti-Scribble antibody as shown in the right panel with approximately 1 mg testis lysate protein per sample for Co-IP, in which anti-Vangl2 antibody or normal goat IgG served as the corresponding positive and negative control. Specificity of the Co-IP shown herein was further confirmed by quantifying the amount of remaining Scribble in the S/N not pulled down in the immunocomplexes by protein A/G Plus to illustrate considerable amount of Scribble was associated with Vangl2 (see right lane in the lower panel immunoprecipitated with a goat anti-Vangl2 antibody, which had considerable less Scribble vs the amount of Scribble in the left lane immunoprecipitated with normal goat IgG, and considerably fewer Scribble was found in the sample immunoprecipitated with a goat anti-Scribble antibody shown in the middle lane; 20 μL S/N from the 250-μL sample was used for this experiment). IgG (see the denoted heavy and chains) or β-actin served as the protein loading control. IB, immunoblotting; IP, immunoprecipitation. D, Co-IP was also performed using lysates of Sertoli cells cultured in vitro with an established TJ-permeability barrier and subjected to overexpression of Vangl2 (pCI-neo/Vangl2) vs Ctrl (transfected with empty pCI-neo vector) in which one of the controls was performed with Co-IP with normal IgG whereas the other was performed with Co-IP with the corresponding immunoprecipitating antibody to assess the Vangl2-partner protein interaction at the basal level (ie, without Vangl2 overexpression). When Vangl2 was overexpressed in Sertoli cells, Vangl2 was found to interact with actin and N-cadherin but not Eps8 and Arp3. β-Actin served as the protein loading control.