FIG 4.

vPK causes phosphorylation of KAP1 at serine 824 during viral lytic activation. (A and B) HH514-16 (A) and CLIX-FZ (B) cells transfected with empty vector (EV) or pFLAG-vPK were treated with NaB (A) or doxycycline (B) 24 h later and harvested at the indicated times posttreatment. Cell lysates were analyzed by immunoblotting with antibodies against p-S824 KAP1, KAP1, and FLAG. (C) LCL was transfected with pLIX-FZ (encoding doxycycline-inducible BZLF1) plus empty vector (EV) or pLIX-FZ plus pFLAG-vPK, treated with doxycycline 24 h later, and harvested after another 24 h for immunoblotting with antibodies to p-S824 KAP1, KAP1, and FLAG. (D) HH514-16 cells were transfected with scrambled siRNA or si-BGLF4, treated with NaB after 8 h, and harvested after another 24 h. (E) LCL transfected with scrambled siRNA plus empty vector, scrambled siRNA plus ZEBRA-expressing plasmid (pHD1013-Z), si-BGLF4 plus empty vector, or si-BGLF4 plus pHD1013-Z were harvested 24 h later. Cell lysates from harvested HH514-16 cells and LCL were immunoblotted with the indicated antibodies. (F) 293-p2089 and 293-G4⁻/G5⁻ cells were transfected with the indicated plasmid combinations (pLIX-FZ, pHD1013-Z, EV, pG5, and pG4) and harvested 24 h later for immunoblotting with the indicated antibodies. All experiments were performed at least 3 times.