BRCA1 Forms a Functional Complex with -H2AX as a Late Response to Genotoxic Stress
Figure 3
H2AX-E139 is ubiquitinated at K118 or K119. (a) Critical amino acids within the C-terminus are shown. (b) HA-tagged ubiquitin (HA-Ub) was transfected into cells with or without H2AX-V5-H6. His-tagged (H6) proteins were purified from chromatin fractions then probed sequentially for the presence of HA-ubiquitin and V5 epitopes (WB). 10% of the unfractionated extract was blotted directly and probed for -actin. (c) H2AX variants containing E139 (to mimic S139 phosphorylation) and various lysine (K) to arginine (R) Substitutions were transfected with HA-tagged ubiquitin. Histidine-tagged proteins were purified from chromatin fractions then probed sequentially for the presence of HA-ubiquitin and V5 epitopes. 10% of the unfractionated extract was blotted directly and probed for -actin. The star (lane 8) indicates the absence of HA-tagged ubiquitin. Arrows indicate unmodified and ubiquitinated H2AX.
