Figure 8  HBV envelopment of nucleocapsids occurs in ERGIC-53- and Sec24A-depleted cells. (A) ERGIC-53 KD, Sec24A KD or BFA-treated cells were transfected with the HBV replicon, processed for IF and stained with L- (57761, L, green) and core-specific (B0586, C, red) antibodies. IF analyses and quantifications were done as above. (B) HuH-7 cells were treated with siCon-, siERGIC-53- or siSec24A-specific duplexes, followed by transfection with pHBV*. Cells were lysed by repetitive freeze-thaw cycles, and intracellular enveloped virions were precipitated with L-specific antibodies in the absence of detergents and assayed by PCR. Error bars indicate the standard deviations from the mean of two experiments measured in duplicates. To monitor HBV protein expression, cell extracts were probed by L- and core-specific WB, as above. A nonspecific band stained by the antisera served as a control for identical gel loading. (C) To probe for IP efficiencies, cells were treated and processed exactly as in B, and immunoprecipitated samples were analyzed by L-specific WB (MA18/7). The p39 and gp42 forms of L are indicated. Experiments were done in duplicate, and representative blots are shown. ** p < 0.01 compared to control.