Fig. 3.

Binding of Insig-1 to Scap does not block binding of antibody to the MELADL sequence. On day 0, Scap-deficient SRD-13A cells were set up in 60-mm dishes. On day 2, cells were transfected with 2 μg of pTK-SREBP-2 (all dishes) and 0.2 μg of pCMV–Scap and 0.05 μg of pCMV-Insig-1-Myc as indicated. Twelve hours after transfection, cells were switched to sterol-depleting medium containing 1% HPCD and incubated at 37°C for 1 h. The cells were then incubated with sterol-depleting medium (without HPCD) in the absence or presence of 1 μg/ml of 25-HC. After incubation for 3 h at 37°C, the cells were harvested, and aliquots of microsomal membranes (150 μg), in a final volume of 0.3 ml of Buffer B, were mixed with 3 μg of anti-MELADL antibody in the absence or presence of 0.5 mg of the MELADL-containing synthetic peptide as indicated. After incubation for 1 h at 4°C, the microsomal membranes were pelleted by centrifugation, solubilized in Buffer D (50 mM Hepes-KOH, pH 7.2/150 mM KCl/1 mM MgCl₂, and a mixture of protease inhibitors; see Materials and Methods) supplemented with 0.1% Nonidet P-40, and incubated with Protein A/G beads for 30 min at 4°C. The resulting supernatant (S) and pellet (P) (10% of S) fractions were subjected to 8% SDS/PAGE and immunoblot analysis with IgG-9D5 (anti-Scap) and IgG-9E10 (anti-Insig-1).