FIGURE 5.

The effects of CideB blockade on VTV formation. VTV budding assays were performed in which hepatic ER containing [³H]TAG was incubated with or without rat hepatic cytosol in the presence of an ATP-regenerating system. After incubation, the reaction mixture was resolved on a continuous sucrose density gradient, and fractions (0.5 ml) were collected. The [³H]TAG dpm values in all fractions (500 μl) were measured. A, prior to budding assay, the ER containing [³H]TAG was incubated at 4 °C for 2 h with preimmune IgG, anti-CideB, or boiled anti-CideB antibodies, and antibodies in each case were removed by washing. Cytosol was pretreated at 4 °C for 2 h with preimmune IgG, anti-CideB, or boiled anti-CideB antibodies bound to agarose beads, and the antibodies were removed by centrifugation. Results are mean ± S.D. (n = 4). Bars labeled with different symbols show p < 0.01 using one-way ANOVA. No cyto, absence of cytosol. B, the ER containing [³H]TAG and the hepatic cytosol similar to panel A were treated prior to performing budding assays, with the indicated antibodies. Data are mean ± S.D. (n = 4). Bars labeled with different symbols are p < 0.005 using one-way ANOVA. C, ER containing [³H]TAG and hepatic cytosol were pretreated with the indicated antibodies and used in VTV budding assays as described above. For details, see “Experimental Procedures.” Values are mean ± S.D. (n = 4). Bars labeled with different symbols have p < 0.001 using one-way ANOVA.