Figure 5.

Recycling assay illustrating the differential fate of the internalized BTB integral membrane proteins after treatment of Sertoli cells with either T or TGF-β2. A) Recycling assay was performed on day 5 after Sertoli cell isolation. After biotinylation of cell surface proteins, Sertoli cells were incubated at 35°C for 2 h to allow endocytosis. Biotin on the uninternalized cell surface proteins was stripped. Cells were then incubated with either T (2×10⁻⁷ M) or TGF-β2 (10 ng/ml) vs. controls (no treatment) at 35°C for various time points to allow recycling of internalized proteins back to the cell surface. The newly appeared biotinylated (and recycled) proteins on the cell surface were obtained by 0.01% trypsin, whereas proteins remaining in the cytosol were collected by IP buffer. B, C) Percentage of internalized and biotinylated proteins remaining in the cytosol over time in the recycling assay in the presence of either T or TGF-β2 vs. controls are shown. The percentage of internalized and biotinylated proteins at time 0 was arbitrarily set at 100. Each bar is the mean ± sd of three independent experiments using different batches of primary Sertoli cell cultures. In each experiment, each time point had triplicate cultures. D, E) Reappearance of internalized and biotinylated occludin and JAM-A on the Sertoli cell surface with or without T was monitored by immunoblotting in three independent experiments with triplicate cultures for each time point in each experiment. The immunoblots (insets; D, E) are results of a representative experiment, illustrating an increase in the level of proteins on cell surface vs. time 0; the presence of T accelerated the kinetics of recycling of JAM-A and occludin. Two additional experiments yielded similar results. The level of protein at time 0 was arbitrarily set at 1. F) Effects of TGF-β2 on the kinetics of recycling of BTB proteins. Reappearance of internalized and biotinylated occludin and JAM-A proteins on Sertoli cell surface was monitored by immunoblotting, illustrating an increase in protein levels on cell surface vs. time 0, but the presence of TGF-β2 significantly reduced the kinetics of recycling of occludin, but not of JAM-A, back to the Sertoli cell surface. The level of protein at time 0 was arbitrarily set at 1, against which statistical analysis was performed. Each bar is the mean ± sd of three independent experiments with triplicate cultures for each time point in each experiment. Statistical analysis was performed by two-way ANOVA followed by Dunnett’s test, comparing between a treatment group vs. its corresponding control (Ctrl) and over time. *P < 0.05; **P < 0.01; ns, not significantly different.