FIGURE 6.

Knockdown of CideB in primary hepatocytes abrogates VTV formation. Primary hepatocytes were transfected with either CideB siRNA (5′CAUGAGCUGCGAUUUUCAATT3′) or control siRNA (Silencer select siRNA, Ambion). ER containing [³H]TAG and cytosol were prepared and purified from both sets of hepatocytes as described under “Experimental Procedures.” A, protein samples of whole cell lysate (WCL). siCideB, CideB siRNA; siControl, control siRNA. B, hepatic ER membranes. C, cytosol prepared from untreated primary hepatocytes or treated with either CideB siRNA (siCideB) or control siRNA (siControl) was probed with specific anti-CideB, anti-β-actin, or anti-calnexin antibodies. D, VTV budding assays were performed using ER containing [³H]TAG and cytosol, which were isolated from primary hepatocytes treated with either CideB siRNA (siCideB) or control siRNA (siControl). As a negative control, ER containing [³H]TAG isolated from untreated hepatocytes was incubated in the absence of cytosol (No cyto). Results are mean ± S.D. (n = 4). Bars labeled with different symbols depict p < 0.001 using one-way ANOVA.