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"To induce KSHV lytic reactivation in 293TBAC16 cells, we transfected the cells with a plasmid expressing 3xFLAG-RTA. . Antibodies The following primary antibodies were used in the study: anti-RTA (gift from Yoshihiro Izumiya, UC Davis), anti-ORF6 (gift from Gary S. Hayward, Johns Hopkins University), anti-ORF45 (Santa Cruz sc-53883), anti-K8.1 (Santa Cruz sc-65446), anti-RNF20 (Cell Signaling 11974S), anti-RNF40 (Cell Signaling 12187S), anti-FLAG (Sigma F1804), anti-HA (BioLegend 901501), anti-V5 (Abcam ab27671), anti-tubulin (Sigma T5326), anti-H2B (Active Motif 39210), anti-H2BK120ub (Cell Signaling 5546S), anti-H3 (Abcam ab1791), anti-H3K4me3 (Active Motif 39159), anti-RNA polymerase II CTD (Cell Signaling 2629), and anti-RNA polymerase II CTD phospho Ser2 (Abcam ab5095). . Plasmids, DNA transfection, and luciferase assays We used pCDH-CMV-MCS-EF1-puro vector to express the different epitope-tagged full-length and mutant versions of RTA, RNF20, and RNF40 in cells."
Figure Legend
"Fig 10 RNF20 regulates RNA polymerase II occupancy on RTA promoter during lytic reactivation of KSHV. (A–D) iBCBL1-3xFLAG-RTA cells were transduced with lenti-shControl or lenti-shRNF20 then reactivated for 12 h, and ChIP-qPCR analysis was performed at the indicated sites of the RTA promoter and gene body. (A) H2BK120ub ChIP. (B) H3K4me3 ChIP. (C) RNA polymerase II (RNAP II) ChIP. (D) ChIP for RNAP II Serine 2 phosphorylation. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3). "