Skip to main content
. 2013 Jan;33(1):111–126. doi: 10.1128/MCB.01024-12

Fig 1.

Fig 1

H2AX is ubiquitylated and sumoylated. (A) H2AX is posttranslationally modified. Empty vector (−) or vector expressing 3×Flag-tagged H2AX (fH2AX) (+) was transfected into U2OS cells, and then H2AX was immunoprecipitated with anti-Flag antibody and detected by Western blotting with this antibody. Arrows indicate full-length fH2AX as well as two, slower-migrating fH2AX species. (B) H2AX is ubiquitylated and sumoylated. As indicated, U2OS cells were transfected with fH2AX or empty vector, either together with (+) or in the absence of (−) vectors encoding HA-tagged ubiquitin (HA-Ub) or SUMO1. Cells were treated as in panel A and then analyzed with the indicated antibodies. α-Flag, anti-Flag. Arrows indicate H2AX species. (C) H2AX is preferentially sumoylated by SUMO1. fH2AX coimmunoprecipitations with either HA-SUMO1 or HA-SUMO3 were performed as in panel B. (D and E) Endogenous H2AX is sumoylated. For panel D, U2OS cells were untreated (unt) or were treated with the indicated DNA-damaging agents and then analyzed and detected by Western blotting with a γH2AX-specific antibody. Etop, etoposide. For panel E, 293T cells were transfected with either an empty vector or a vector encoding HA-tagged SUMO1. Cells were untreated or treated with 5 Gy IR and then analyzed and detected by Western blotting with the specified antibodies. Arrows indicate the various H2AX species. Note the shift of mobility of the H2AX sumoylation band in cells expressing HA-tagged SUMO1. WCE, whole-cell extracts. (F) H2AX sumoylation is dependent on PIAS4. U2OS cells were transfected with control (siLuc), PIAS1, or PIAS4 siRNAs followed by transfection with both fH2AX and HA-tagged SUMO1. Cells were analyzed as in panel B. Asterisks indicate background bands.