Figure 3. RNAi-mediated knockdown of PDP1 enhances GGpp-induced ERAD of HMG CoA reductase.

SV-589 cells were set up on day 0 at a density of 1.5 × 105 cells per 60 mm dish in medium A supplemented with 5 % FCS. On day 1 cells were transfected in medium A containing 5 % FCS with siRNAs targeting mRNAs that encode GFP or PDP1. In (A), cells were transfected with PDP1 siRNAs A–E. (A) On day 3, cells were harvested, lysed, and aliquots of resulting whole-cell lysates (10 µg protein loaded/lane), followed by immunoblot analysis using anti-PDP1, anti-calnexin, and IgG-A9 (against reductase). (B) On day 3, cells were switched to medium A supplemented with 5 % FCS in the absence or presence of 10 µM compactin. Following incubation for 24 hr at 37 °C, cells were refed the identical medium and the indicated concentration of GGOH and incubated for an additional 24 hr. Cells were then harvested for subcellular fractionation; aliquots of resulting membrane fractions (15 µg protein loaded/lane) were subjected to SDS-PAGE and immunoblot analysis as described in (A). The amount of reductase was determined by quantifying the band corresponding to reductase using ImageJ software. Each value represents the amount of reductase protein relative to that in untreated cells transfected with GFP siRNA, which was arbitrarily set as 1.

Figure 3—figure supplement 1. Knockdown of PDP1 enhances proteasome-mediated ERAD of HMG CoA reductase.

SV-589 cells were set up on day 0 at a density of 1.5 × 105 cells per 60 mm dish in medium A supplemented with 5 % FCS. On day 1 cells were transfected in medium A containing 5 % FCS with siRNAs targeting mRNAs that encode GFP or PDP1. On day 3, cells were treated in the absence or presence of 10 µM compactin and the indicated concentration of GGOH. In (A), cells were also treated with 10 µM cycloheximide and in (B), some of the cells received 10 µM MG-132. Following incubation for 24 (A) or 6 hr (B) at 37 °C, cells cells were harvested for subcellular fractionation; aliquots of resulting membrane fractions (15 µg protein loaded/lane) were subjected to SDS-PAGE followed by immunoblot analysis using anti-PDP1, anti-calnexin, and IgG-A9 (against reductase).