Figure 4. RNAi-mediated knockdown of PDP1 abolishes compactin-induced, Golgi-to-ER redistribution of UBIAD1 and stabilization of HMG CoA reductase.

(A) Proposed role of PDP1 in modulating levels of GGpp that stimulate transport of UBIAD1 from membranes of the ER to Golgi. (B) SV-589/pMyc-UBIAD1 cells were set up on day 0 at 7.5 × 10⁴ cells per well of 6-well plates with glass coverslips in medium A supplemented with 5 % FCS. On day 1, the cells were transfected in identical medium with siRNAs against GFP or PDP1 mRNAs as described in the legend to Figure 3. Following incubation for 16 h at 37 °C, cells were switched to medium A supplemented with 5 % FCS in the absence or presence 10 µM compactin. After 2 hr, cells were fixed, permeabilized, and analyzed by immunofluorescence microscopy using IgG-9E10 (against Myc-UBIAD1) as described in the legend to Figure 2. (C–E) SV-589 cells were set up on day 0 at 2 × 10⁵ cells per 60 mm dish in medium A containing 5 % FCS. On day 1, cells were transfected in identical medium with siRNAs against GFP or PDP1. On day 2, cells were refed medium A supplemented with either 5 % FCS (C) or 10 % LPDS (D and E). Following incubation for 16 hr at 37 °C, the cells were treated with 10 µM compactin in the absence (C and D) or presence (E) of 1 µg/ml 25-HC. The cells were then incubated for the indicated period of time, after which detergent lysates were prepared and subjected to immunoblot analysis using IgG-A9 (against reductase), anti-PDP1, and anti-calnexin.