Figure 8.

A study by Co-IP to assess changes in the interactions between actin- and MT-based binding and regulatory proteins with the corresponding cytoskeleton during the release of germ cells from the testis in the adjudin model. Lysates of testes (1 mg protein in each Co-IP reaction) from rats were obtained and used for Co-IP (as described in Materials and Methods) following treatment with adjudin (50 mg/kg b.w., oral gavage) at 6, 12, 24, and 96 hours vs 0 hours (control) with n = 3 rats per time point including control. (a) Actin regulatory proteins: Eps8 (an actin barbed-end capping and bundling protein causing actin filaments to assume a bundled configuration as those found at the ES), formin 1 (an actin nucleation protein capable of generating long stretches of actin microfilaments), and Arp3 (a branched actin polymerization protein causing linear actin filaments to become a branched network) were used for this study. Also shown is immunoprecipitated β-actin, which served as the positive control. Uncropped gel images shown herein can be found in Supplemental Fig. 5 ^{(3.3MB, pdf)} . (b) MT regulatory proteins: EB1 (a +TIP protein known to stabilize MTs) and MARK4 (a Ser/Thr protein kinase known to induce MT catastrophe) were also used for this study. Also shown is the immunoprecipitated α-tubulin, which was confirmed by using an anti–β-tubulin antibody for immunoblotting (IB) (see Table 1), which served as the positive control. Changes in protein-protein interaction of these selected regulatory proteins with the corresponding β-actin and α-tubulin, the building blocks of actin- and MT-based cytoskeletons, were assessed by Co-IP. In short, anti-actin IgG or anti–α-tubulin IgG, serving as the immunoprecipitating antibody, was incubated with testis lysates obtained from rats treated with adjudin for 6, 12, 24, and 96 hours vs 0 hours (control). Thereafter, immunocomplexes were pulled out by Protein A/G Plus-agarose beads, the interacting proteins with either actin or α-tubulin were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the interacting proteins (and their changes following adjudin treatment) with either actin or MT were visualized by immunoblottings using corresponding specific antibodies (Table 1). Uncropped gel images shown herein can be found in Supplemental Fig. 6 ^{(3.3MB, pdf)} . The steady state of the regulatory proteins (i.e., Eps8, formin 1, Arp3, EB1, and MARK4) in testis lysates following adjudin treatment (without Co-IP) was also assessed and annotated as “Lysate.” Representative immunoblot data were shown in the upper panel in either (a) or (b), and the histograms below summarize results of these findings with n = 3 independent experiments using testes from different rats. *P < 0.05 by ANOVA.