FIGURE 6.
TRPP2 is subject to lysosomal degradation. A, drug incubation of HeLa cells started 24 h after wild-type TRPP2 transfection. Small molecule inhibitors were added to cell medium, 200 μm chloroquine (C) for 24 h, 210 μm MG-132 (M) for 6 h, or 6 mm ammonium chloride (A) for 24 h. As a negative control, untreated (ø) cells were processed in parallel. Cells were counted after harvesting, and sample volume was normalized for cell count. β-Actin was used as the Western blot (WB) loading control. Wild-type TRPP2 protein expression in cells without drug treatment was set to 100%, and relative TRPP2 amounts after drug treatment were calculated. Wild-type TRPP2 was significantly more abundant in cells treated with chloroquine compared with control cells (mean = 575.36%, n = 4, p = 0.0003). B, likewise, TRPP2Δ5-Glyc protein levels increased in the presence of chloroquine (mean, 329.64%; n = 4; p = 0.048). C, incubation of wild-type TRPP2 (mean, 203.07%; n = 3; p = 0.04) and TRPP2Δ5-Glyc (mean, 218.69%; n = 3; p = 0.02) with ammonium chloride significantly increased protein levels compared with untreated controls.