Figure 7

A study to assess the effect of Abcb1b single knockdown on the fate of endocytosed BTB integral membrane proteins induced by F5-peptide in Sertoli cell epithelium. (a) Changes in the distribution of proteins at the Sertoli–Sertoli cell interface were examined by immunofluorescence microscopy. Junction proteins, including occludin, ZO-1, N-cadherin and β-catenin, relocalized back to the cell surface at 12 h after F5-peptide removal in the scramble silencing group. However, cells with Abcb1b knockdown displayed a mislocalization of these junction proteins. Micrographs in the fourth row are the corresponding grayscale images of the true-color images in the third row to better depict changes in protein localization, scale bars=20 μm, which applies to all micrographs. (b–d) Recycling assay illustrated the fate of the internalized BTB integral membrane proteins induced by F5-peptide in Src or Abcb1b RNAi treated Sertoli cells. 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 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) Immunoblots and percentage of internalized and biotinylated proteins remaining in the cytosol over time in the recycling assay were shown. The percentage of internalized and biotinylated proteins at time 0 was arbitrarily set at 100. Each bar is the mean±S.D. of three independent experiments using different batches of primary Sertoli cell cultures. In each experiment, each time point had triplicate cultures. (c,d) Reappearance of internalized and biotinylated occludin and N-cadherin on the Sertoli cell surface after Scr or Abcb1b RNAi was monitored by immunoblotting (the insets) in three independent experiments with triplicate cultures for each time point in each experiment. The level of protein at time 0 was arbitrarily set at 1. Statistical analysis was performed by two-way ANOVA followed by Dunnett’s test, comparing between Abcb1b RNAi group versus its corresponding Scr RNAi group and over time. *P<0.05; **P<0.01