Figure 5.

Effects of CD82 and its cholesterol binding on ERM proteins, the plasma membrane–actin cytoskeleton connectors. (a) Western blot analysis of total and phosphorylated ERM (Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)) proteins in Du 145 transfectant cells. For cell lysis in suspension, the cells were detached and spun down, and cell pellets were lysed with RIPA buffer. For cell lysis in situ, the cells attached to culture dishes were directly lysed with RIPA buffer. (b) Total and phosphorylated ERM (Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)) proteins in PC3, HT1080, and LnCap transfectants were examined with Western blot. The cells were lysed directly in culture dishes. (c) Regulation of Ezrin distribution by CD82 or its cholesterol binding. Cells cultured on glass coverslips for 4–5 days were fixed, permeabilised and incubated with Ezrin Ab and the secondary Ab, phalloidin, and DAPI, and examined with confocal microscopy. Scale bar: 10 µm. (d) Releases of ERM proteins via EVs. Exosomes isolated from culture supernatants of the cells were lysed with RIPA buffer, equal amounts of EV proteins (5 μg/lane) were loaded to SDS–PAGE, and ERM and CD9 proteins were examined in Western blot. (e)–(f) The analyses were performed as described in (c). (g) Du145-CD82 WT transfectant cells were cultured on glass coverslips in the serum-free DMEM containing either DMSO (0.1% v/v), TAK-475 (20 µM) or cholesterol (15 µg/ml)/BSA(2%) for 48 h, fixed, stained for Ezrin and F-actin, and photographed with confocal microscopy. Scale bar: 10 µm. Extracellular Ezrin proteins were quantified with ImageJ and presented as relative fluorescence intensity (mean ± SEM, n = 3 individual experiments). *: p < 0.05, **: p < 0.01 and ***: p < 0.001. (h) Cells were treated with DMSO (0.1% v/v) or TAK-475 (20 µM) in serum-free DMEM for 48 h, detached with 2 mM EDTA/PBS and lysed with RIPA buffer. Cell lysates were examined for Ezrin and actin in Western blot.