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. 2015 Oct 27;291(2):959–967. doi: 10.1074/jbc.M114.624478

FIGURE 3.

FIGURE 3.

USP11 is a unique deubiquitin enzyme for γH2AX. A, HeLa cells were transfected with pool of siRNA oligonucleotides of the positive DUBs by the assay in Fig. 2. Then HeLa cells were transiently transfected with plasmids DNA expressing Flag-H2AX, HA-RNF8, and His-UB. The whole cell extracts and the elution of Ni-NTA-agarose bead pulldown were assayed by Western blot analysis using antibodies against γH2AX, H2AX, Flag, and HA. B, HeLa cells were transfected with a pool of siRNA oligonucleotides of DUBs, treated with 12 grays γ-irradiation, and recovered for 30 min. The whole cell extracts were assayed by Western blot analysis using antibodies against γH2AX, H2AX, USP11, and actin. C, HeLa cells were transfected with different siRNA oligonucleotides of USP11 and then treated with 12 grays γ-irradiation and recovered for 30 min. The whole cell extracts were assayed by Western blot analysis using antibodies against γH2AX, H2AX, USP11, and actin. D, HeLa cells were transfected with different siRNA oligonucleotides of USP11 and then transiently transfected with plasmids DNA expressing Flag-H2AX, HA-RNF8, and His-UB. The whole cell extracts and the elution of Ni-NTA-agarose bead pulldown were assayed by Western blot analysis using antibodies against γH2AX, H2AX, Flag, and HA. E, 293 cells were transiently transfected with plasmids DNA expressing Flag-H2AX, HA-RNF8, His-UB, and Flag-USP11 wild type or Flag-USP11 C318A inactive mutant. The whole cell extracts and elution of Ni-NTA-agarose bead pulldown were assayed by Western blot analysis using antibodies against γH2AX, H2AX, Flag, and HA.