Figure 4: H2Bv3 is ADP-ribosylated at E18 and E19 in vivo.
From: Site-specific ADP-ribosylation of histone H2B in response to DNA double strand breaks
(A) Gene replacement strategy to replace the endogenous h2b locus with either wild-type h2b (h2bwt), or h2b containing E18AE19A mutations (h2bE18AE19A). (B and C) Two independent h2bwt and h2bE18AE19A strains were left untreated or exposed to 300 μg/ml phleomycin for 60 minutes as indicated. Histone-enriched acid extracts were prepared and western blotting performed using the indicated antibodies. Arrow heads indicate the position of ADP-ribosylated H2B protein in h2bwt cells. (D) Wild-type H2Bv3-Flag or H2Bv3-Flag with the indicated mutations were expressed in Ax2 cells. Whole cell or chromatin extracts were prepared and western blotting performed with the indicated antibodies. (E) Ax2 cells expressing wild-type H2Bv3-Flag or the indicated H2Bv3-Flag mutants were left untreated or exposed to 300 μg/ml of phleomycin for 1 hour as indicated. Histone-enriched acid extracts were prepared and western blotting performed with the indicated antibodies. ADP-ribosylated H2Bv3-Flag is indicated with an arrow.
