Fig. 3.

Inhibition of sterol-induced ubiquitination of HMG CoA reductase by RNA interference-mediated knockdown of Trc8 and gp78. SV-589 cells were set up on day 0, transfected with the indicated siRNAs on days 1 and 2, and depleted of sterols as described in Fig. 2A. Sterol-depleted cells were subsequently incubated for 30 min in medium B containing 10% LPDS, 50 μM compactin, and 10 μM MG-132 in the absence or presence of 1 μg/mL 25-HC plus 10 mM mevalonate as indicated. Cells were harvested and immunoprecipitated with polyclonal anti-reductase, followed by immunoblot analysis with IgG-A9 (against reductase) and IgG-P4D1 (against ubiquitin). Total RNA from 25-HC plus mevalonate-treated cells in (A) and (C) was subjected to first-strand cDNA synthesis and real-time PCR. Each value for cells transfected with the indicated siRNA represents the amount of the indicated mRNA relative to that in control cells transfected with the GFP siRNA.