Figure 4.

Brefeldin A induces endo H-resistance of SCAP in the presence of sterols. On day 0, CHO-7 cells were set up in medium A supplemented with 10% FCS. On day 2, cells were switched to medium A containing 10% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 10.5 hr, cultures received either no addition (lanes 1–16) or 5 μg/ml swainsonine (lane 17). After a 30-min preincubation, cells were incubated for an additional 5 hr with 0.1% (vol/vol) methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. Cells were harvested, and membrane fractions were prepared as described in Materials and Methods. Aliquots (54 μg of protein) were incubated in the absence (lanes 1–4) or presence (lanes 5–17) of trypsin. Proteolysis was stopped, and the samples were incubated either in the absence (lanes 1–8) or presence (lanes 9–17) of the indicated glycosidase, subjected to SDS/PAGE (5–12% gel), and transferred to nitrocellulose. The filter was blotted with 10 μg/ml IgG-9D5 (anti-SCAP) and exposed to film for 2 min.