Figure 4. Sterol-mediated regulation of HMGCR in mouse embryonic fibroblasts (MEFs) from WT and Ubiad1^Ki/Ki mice.

MEFs from WT and Ubiad1^Ki/Ki mice were set up for experiments on day 0 at 2 × 10⁵ cells per 10 cm dish in MEF medium supplemented with 10% fetal calf serum (FCS). (A) On day 3, cells were harvested for subcellular fractionation. Aliquots of resulting membrane and nuclear extract fractions (35–50 µg total protein/lane) were subjected to SDS-PAGE, followed by immunoblot analysis using antibodies against the indicated proteins. (B) On day 3, cells were harvested for measurement of Hmgcr mRNA levels by quantitative RT-PCR and total cholesterol levels using a colorimetric assay as described in ‘Materials and methods.’ (C and D) On day 2, cells were depleted of isoprenoids through incubation for 16 hr at 37°C in MEF medium containing 10% lipoprotein-deficient serum, 10 µM sodium compactin, and 50 µM sodium mevalonate. The cells were subsequently treated with 1 µg/ml 25-HC as indicated; in (D), the cells also received 10 µM MG-132. (C) After 4 hr at 37°C, cells were harvested for preparation of membrane and nuclear extract fractions (35–50 µg total protein/lane) that were analyzed by immunoblot with antibodies against the indicated protein. (D) Following incubation for 1 hr at 37°C, cells were harvested, lysed in detergent-containing buffer, and immunoprecipitated with 30 µg polyclonal anti-HMGCR antibodies. Immunoprecipitated material was subjected to SDS-PAGE and immunoblot analysis with IgG-A9 (against HMGCR) and IgG-P4D1 (against ubiquitin).