(A) 53BP1-GFP transfected U2OS cells were damaged by laser irradiation of the indicated nuclear region. The dynamics of 53BP1-GFP during the DNA damage response on control and SETD2-depleted cells was monitored by live cell imaging for 15 min after laser irradiation. One representative experiment from over 50 individual cells recorded is shown. (B) Control and SETD2 RNAi-depleted U2OS cells were transfected with a 53BP1-GFP expression plasmid. Western blot analysis was performed with antibodies against GFP (to detect 53BP1-GFP), H3K36me3, total histone H3, and α-tubulin. Molecular weight markers (KDa) are shown on the left. (C) SETD2-GFP transfected U2OS cells were lysed and processed for western blot with antibodies against GFP (to reveal SETD2-GFP), H3K36me3 and α-tubulin. Molecular weight markers (KDa) are shown on the left. (D) Live-cell images of SETD2-GFP dynamics were recorded upon laser-induced DNA damage of the indicated nuclear region of U2OS cells during 15 min (E) H3K36me3 was immunoprecipitated from MNase-digested U2OS cell extracts and each of the four nucleosomal histones was detected by immunoblotting of the SDS-PAGE resolved complexes. (F) Nuclear co-immunoprecipitations before and 6 hr after NCS treatment were performed on U2OS cells using the indicated antibodies (I.P.: 53BP1 and H3K36me3). The Input lane represents total cell lysates and (−) denotes the negative control immunoprecipitation (beads only). Purified complexes were resolved by SDS-PAGE and blotted with antibodies against γH2AX or H3K36me3.