Figure 6.

A study by immunofluorescent microscopy and immunoblotting to demonstrate the targeting of BTB integral membrane proteins into late endosomes for degradation by TGF-β2. A) Sertoli cells were incubated with either T or TGF-β2 vs. controls (no treatments) at 35°C for 3 h to allow endocytosis and recycling of BTB proteins. Cells were then fixed and stained with anti-occludin and anti-Rab 9 (a late endosome marker) antibody to study the fate of the internalized proteins. After treatment of Sertoli cells with TGF-β2, the steady-state protein level of occludin (green fluorescence, FITC) at the cell-cell interface was much weaker (i vs. a, d; e, h), with the presence of more internalized occludin vesicles in the cytosol and an increase in Rab 9 staining (red fluorescence, Cy3) (j vs. b, f). The internalized occludin vesicles were found to colocalize with Rab 9 when cells were treated with TGF-β2 but not with T (arrowheads), shown in the magnified images in m and n from the boxed areas in l. Scale bars = 10 µm (a–l); 3 µm (m, n). B) Immunoblots illustrating the increase in the protein level of Rab 9 when cells were treated with TGF-β2 but not with T or in the control (Ctrl) cells. C) Statistical analysis was performed by comparing the protein level of Rab 9 when Sertoli cells were treated with T or TGF-β2 vs. controls. Protein at time 0 in the control group was arbitrarily set as 1, against which statistical analysis was performed. Each bar is the mean ± sd of three different experiments. Statistical analysis was performed by two-way ANOVA followed by Dunnett’s test, comparing each treatment group vs. its corresponding control group over time. *P < 0.05; **P < 0.01; ns, not significantly different. D) Immunoblot analysis of lysates (~50 µg of total protein/lane) of seminiferous tubules (ST) and Sertoli cells (SC) using the mouse anti-Rab 9 antibody.