Figure 3.

Specificity of the sterol-regulated oligosaccharide modifications of SCAP. 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 16 hr, cells were harvested, and membrane fractions were prepared as described in Materials and Methods. (A) Aliquots (60 μg of protein) were incubated in the absence or presence of trypsin as indicated. Proteolysis was stopped, and the samples were incubated either in the absence (lanes 1–4) or presence (lanes 5–10) 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 25 sec. (B) Aliquots of the same membrane fractions used in A (16 μg of protein) were incubated without prior trypsin treatment either in the absence (lanes 1, 2, 9, and 10) or presence (lanes 3–8) of the indicated glycosidase, subjected to SDS/PAGE (6.5% gel), and transferred to nitrocellulose. Filters were blotted with 2 μg/ml anti-BiP antibody (Upper) and 1 μg/ml antitransferrin receptor antibody (Lower) and exposed to film for 5 sec and 2 sec, respectively. En. H, endo H; PN. F, PNGase F; Neur., neuraminidase; TfR, transferrin receptor.