FIG 6.

PJA1 facilitates the binding of the SMC5/6 complex to viral and episomal DNAs. (A and B) 293T cells were plated in 6-cm dishes and cotransfected with 3 μg CMV-Luc and then with 2 μg pFlag-NSE1 (A) or 2 μg pcDNA3.1-PJA1B (B). NSE4 binding of the episomal reporter or GAPDH gene was monitored by ChIP assays using anti-NSE4 antibody. The level of immunoprecipitated DNA was determined by RT-qPCR. (C) 293T cells were plated in 6-well plates and cotransfected with 0.5 μg pCAGGS-HA-NSE4 and 0.5 μg pFlag-SMC5, pFlag-NSE1, or pFlag-NSE3 with or without 0.5 μg pcDNA3.1-PJA1ΔR. (D) 293T cells were plated in 6-well plates and cotransfected with 0.5 μg pCAGGS-HA-NSE4 and 0.5 μg pFlag-NSE1 and then with pcDNA3.1-PJA1BΔR at different concentrations (0, 0.5, and 1.5 μg). Cells were lysed in RIPA lysis buffer. The immunoprecipitates and whole-cell lysates were analyzed by Western blotting with anti-HA or anti-Flag antibody. (E and F) HepG2 cells stably expressing PJA1B were plated in 6-cm dishes and infected with HSV-1 at an MOI of 10 for 8 h. The binding of NSE4 to the VP16 gene promoter (E) or UL36 gene (F) of HSV-1 was monitored by a ChIP assay using beads only, rabbit IgG, or anti-NSE4 antibody. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001.