Loss of SCAP requires ER exit and is blocked by ammonium chloride.
A, CHO-7 cells were set up on day 0 at 1.5 × 106 cells/100-mm dish in medium A supplemented with 5% (v/v) FCS. On day 1, cells were refed medium C containing PF-429242 (50 μm) and fatostatin (10 μm) as indicated. B, CHO-7 cells were set up on day 0 at 1.5 × 106 cells/100-mm dish in medium A supplemented with 5% (v/v) FCS. On day 1, cells were refed medium C containing vehicle, PF-429242 (50 μm), MG132 (50 μm), and NH4Cl (50 mm) as indicated. C, MEFs (wild-type control and Atg7−/−) were set up for experiments on day 0 at 1.5 × 106 cells/100-mm dish in medium D supplemented with 10% (v/v) FCS. On day 1, the cells were refed medium E in the absence or presence of PF-429242 (50 μm). A–C, after 16 h, the cells were fractionated, and membrane fractions (25 μg protein/lane) were subjected to immunoblot analysis using the indicated antibodies.