Figure 3.

The E3 activity–deficient RNF145 blocks sterol-induced ubiquitination and degradation of HMGCR. A, CHO cells were transfected with indicated plasmids, depleted of sterol, and treated with 10 μm MG132 in the presence or absence of 1 μg/ml 25-HC and 10 mm mevalonate for 3 h. Cells were harvested, and lysates were immunoprecipitated with anti-FLAG beads. Input and pellet fractions were immunoblotted (IB) with anti-HA, anti-FLAG, and polyclonal anti-RNF145 antibodies. Results shown are representative of two independent experiments. B, CHO cells were transfected with indicated plasmids, depleted of sterol, and treated with or without 1 μg/ml 25-HC plus 10 mm mevalonate for 5 h. Cells were harvested and subjected to SDS-PAGE and immunoblot analysis. Results shown are representative of two independent experiments.