FIGURE 3:

gp78 is not required for endogenous HMGCR degradation in Rat-1 cells. (A) Rat-1 cells with a stably integrated doxycycline-inducible gp78 shRNA (1831gp78) were stably transfected with Insig-1–Myc. Cells were treated with doxycycline (Dox) for 48 h to induce shRNA expression and maintained in LPDS medium containing compactin and a low concentration of MVA (medium B) for 16 h before assessment of cell lysates. Samples were immunoblotted (IB) as indicated. Duplicate samples are shown. By densitometry, gp78 levels were estimated to be reduced by ∼85% in the doxycycline-treated cells. (B) Cells described in A were treated with or without Dox as in A and incubated in medium B for 16 h. Stability of Insig-1–Myc was assessed by ³⁵S pulse chase and immunoprecipitation with Myc antibodies. (C) Quantification of ³⁵S pulse-chase experiment in B. (D) 1831gp78 or control (CTL) shRNA Rat-1 cells (not transfected with Insig-1–Myc) were treated with Dox and incubated in medium B, as in A. HMGCR degradation was analyzed by ³⁵S pulse chase and immunoprecipitation with HMGCR antibodies. (E) Quantification of ³⁵S pulse-chase experiment shown in D. Inset, relative gp78 levels in this experiment assessed by immunoblotting of cell lysates. gp78 was estimated to be decreased by ∼85% upon knockdown. Note: Control (CTL) shRNA samples shown in D and quantified in E were published previously as controls (Wang et al., 2009).