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. 2012 Sep 24;7(9):e45749. doi: 10.1371/journal.pone.0045749

Figure 2. The interactions between VP16, ICP22 and P-TEFb in vivo.

Figure 2

(A) Co-immunoprecipitation of interaction between ICP22 and VP16 in vivo. CHO-K1 cells were co-transfected with pcDNA-ICP22 and pcDNA-VP16 plasmids, or Hep-2 cells were infected with HSV-1 (MOI = 1) for 40 h. In each case the cell lysates were immunoprecipitated with mouse anti-ICP22 or rabbit anti-VP16 polyclonal antibody, or mouse/rabbit IgG as a control, and immunoblotted with ICP22 or VP16 polyclonal antibody. (B) Co-immunoprecipitation of interactions between ICP22 and P-TEFb, VP16 and P-TEFb in vivo. CHO-K1 cells were transfected with pcDNA-ICP22 and pcDNA-VP16 plasmids, or Hep-2 cells were infected with HSV-1 (MOI = 1) for 40 h. In each case the cell lysates were immunoprecipitated with rabbit anti-Cdk9 and anti-CyclinT1 polyclonal antibodies (rabbit IgG as a control) and immunoblotted with ICP22 or VP16 polyclonal antibody. (C) Co-immunoprecipitation of interaction between ICP22 and VP16 via immunodepletion of P-TEFb. CHO-K1 cells were co-transfected with pcDNA-ICP22 and pcDNA-VP16 plasmids for 40 h. The cell lysates were immunodepleted with Cdk9 and CyclinT1 antibodies or rabbit IgG (control) before following procedure, the equal amounts of the immunodepleted cell lysates immunoblotted with the indicated antibodies (left panel) or immunoprecipitated with anti-VP16 beads followed by immunoblotting with the indicated antibodies (right panel). (D) The effects of Cdk9 and CyclinT1 RNA interference analyzed by western blotting and co-immunoprecipitation of interaction between ICP22 and VP16 via knocking down P-TEFb. Hep-2 cells were transfected for 40 h with si-Cdk9, si-CyclinT1 or the scrambled interfering RNA as a negative control at a concentration of 100 nM. Equal amounts of the cell lysates were immunoblotted with Cdk9, CyclinT1 or GAPDH polyclonal antibodies (upper panel). Hep-2 cells were co-transfected with the specific Cdk9 and CyclinT1 siRNA or the scrambled negative siRNA (100 nM) for 12 h, then infected with HSV-1 (MOI = 1). At 36 h after infection, equivalent amounts of protein from the whole cell lysates were immunoprecipitated with the rabbit anti-VP16 polyclonal antibody or rabbit IgG as a control, and immunoblotted with the ICP22 polyclonal antibody (lower panel).