FIG 2.

Viral entry and transcription are required for down-modulation of p62 during HSV-1 infection. (A) HEL cells were infected with HSV-1(F) (10 PFU/cell) that had been exposed to UV light (1,200 μJ/cm²) for 0, 2, 5, or 15 min, and whole-cell lysates were collected at 24 h postinfection. The levels of p62 protein were analyzed in equal amounts of cell lysates using an anti-p62 antibody. ICP0 expression served as a positive control for infection. β-Actin was used as a loading control. (B) HEL cells were infected with HSV-1(F) at 5 PFU/cell (lane 2) or 10 PFU/cell (lane 3) or with viral stock (10 PFU/cell) that had been heated at 56°C for 5, 10, or 15 min (lanes 4 to 6). Whole-cell lysates were collected at 24 h postinfection, and equal amounts of protein were analyzed by immunoblot analysis, using antibodies against p62 and ICP0. β-Actin served as a loading control. (C) HEL cells were infected with HSV-1(F) (10 PFU/cell) and were left untreated or treated with actinomycin D (ActD; 10 μg/ml) at 1 h postinfection. Whole-cell lysates were collected at 9 h postinfection, and equal amounts of protein were analyzed by immunoblot analysis, using antibodies against p62 or ICP0. β-Actin served as a loading control. (D) The experiment whose results are shown was done as described for panel C except that the cells were treated with cycloheximide (CHX; 100 μg/ml), added at the moment of infection. The cells were harvested at 9 and 24 h postinfection, and equal amounts of proteins were analyzed as described above. (E) HEL cells were infected with HSV-1(F) (10 PFU/cell) and were left untreated or treated with ionomycin (10 μM) or BAPTA.AM (10 μM) at 1 h postinfection. Whole-cell lysates were collected at 9 h after infection, and equal amounts of protein were analyzed by immunoblot analysis, using antibodies against p62 or ICP0. β-Actin served as a loading control. All experiments were repeated three independent times, and representative Western blots are presented.