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. 2016 Jun 30;6:28589. doi: 10.1038/srep28589

Figure 4. Characterization of the binding partners of CRB3 at the BTB by co-immunoprecipitation (Co-IP).

Figure 4

(A) A study by Co-IP to assess protein-protein interactions between CRB3 and selected BTB-associated proteins, such as CRB3-associated protein (e.g., PALS1), TJ proteins (e.g., occludin, ZO-1), basal ES protein (e.g., N-cadherin), actin binding/regulatory proteins (e.g., Arp3, Eps8, palladin), and cytoskeletal proteins (e.g., actin, vimentin) at the BTB using corresponding primary antibodies (Table 1) and lysates from adult rat testes for immunoprecipitation. Rabbit or mouse IgG served as a negative control for the Co-IP experiment, whereas normal testis lysate without Co-IP served as a positive control. The heavy and light chains of IgG served as loading control. (+), positive interaction; (−), negative (or no) interaction. (B) Results of a Co-IP experiment using CRB3 antibody as the immunoprecipitating antibody to confirm interactions between CRB3 and its binding partners shown in (A). Normal rabbit IgG was used for Co-IP and served as a negative control, whereas normal testis lysate without Co-IP served as a positive control. The heavy chain of IgG served as a loading control. (C) Results of Co-IP to confirm interactions between N-cadherin and Arp3 with CRB3 using lysates from Sertoli cells. (D) A study to confirm the efficacy of the Co-IP in which a primary antibody was assessed for its ability to pull out itself vs. normal IgG of the same species which served as the corresponding control. It was noted that the primary antibodies used for Co-IP efficiently pulled down the corresponding target proteins since these proteins were considerably reduced in the supernatant when compared with the control immunoprecipitated with normal IgG of the same species. GAPDH in the supernatant confirmed equal protein loading between samples for Co-IP. Rb, rabbit; Ms, mouse; N-Cad, N-cadherin. Data shown herein are representative findings of a representative experiment from n = 3 independent experiments with similar results.