Fig. 4.

A study by Co-IP to assess the effects of FAK knockdown on occludin–ZO-1 association and the phosphorylation status of occludin in Sertoli cells. On day 4, Sertoli cells cultured at 0.4 × 10⁶ cells/cm² on Matrigel-coated dishes were transfected with nontargeting (Ctrl) or FAK-specific siRNA duplexes for 24 h and were harvested 48 h thereafter. (A) Sertoli cell lysates (≈250 μg protein) were subjected to Co-IP with an anti-occludin antibody, and the blots were probed with anti-occludin or anti-ZO-1 antibodies. Co-IP with rabbit (Rb) IgG served as the negative control (Left). These samples were also used for immunoblotting to illustrate that the FAK knockdown reduced the levels of FAK but not occludin and ZO-1 (Right). (B) Densitometric analyses of Co-IP data of n = 3 with the protein–protein association in controls arbitrarily set at 1. (C) Sertoli cell lysates (≈250 μg of protein) were subjected to Co-IP with an anti-occludin antibody to immunoprecipitate occludin, which was then used for immunoblot analysis by probing with anti-phospho-Tyr, -Ser, or -Thr antibodies. Blots were re-probed with an anti-occludin antibody to confirm equal protein loading. (D) Densitometric analyses of composite data normalized against the occludin level, with the target proteins in controls arbitrarily set at 1. Each bar is the mean ± SD of 3 to 4 experiments. *P < 0.05; **P < 0.01 by Student's t test.