Ubiquitylation and sumoylation site mapping in H2AX by fH2AX-9K to R analysis. (A) Diagram of the fH2AX-9K to R construct. All lysine (K)-to-arginine (R) substitutions in fH2AX-9K to R are indicated. (B) fH2AX-9K to R blocks ubiquitylation and sumoylation. The indicated constructs were transfected into U2OS cells and analyzed as in Fig. 1A. Vec, empty vector. (C) Lysines 118 and 119 of H2AX are the preferred ubiquitylation sites. Cells were transfected with various Flag-H2AX constructs and HA-tagged SUMO1 as indicated. Experiments were analyzed as in panel B with the indicated antibodies, except that anti-Flag immunoprecipitants were eluted with Flag peptide as described in Materials and Methods. Exp., exposure.