Figure 5.

Regulated endo H sensitivity of SCAP is perturbed in 25-hydroxycholesterol-resistant CHO cell lines that harbor mutations in SCAP or SREBP-2. On day 0, the indicated cells were set up in medium A supplemented with 10% fetal calf serum. On day 2, cells were switched to medium A containing 10% lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in either the absence (− sterols) or presence (+ sterols) of 1 μg/ml of 25-hydroxycholesterol plus 10 μg/ml of cholesterol as indicated. After incubation for 16 h, cells were harvested, and membrane fractions were prepared as described in Materials and Methods. Aliquots of membranes (A, 50 μg and B, 54 μg) were incubated in the absence or presence of 17 μg/ml of trypsin as indicated. Proteolysis was stopped, and the samples were incubated at 37°C for 16 h either in the absence (lanes 1–12) or presence (lanes 13–18) of endo H, subjected to SDS/PAGE, and transferred to nitrocellulose. Filters were blotted with 10 μg/ml of IgG-9D5 and exposed to film for 2 min (A) and 20 sec (B). ∗ denotes a cross-reacting protein of unknown identity. Numbers 1–3 on the right denote differentially N-glycosylated forms of the protease-resistant SCAP fragment as described in the legend to Fig. 4.