Figure 2
Figure 2. p30 protein relocalizes on DNA-damage–induced DSBs. (A) Etoposide induces DNA DSBs. HeLa cells were left untreated or treated with etoposide for 24 hours, followed by SDS-PAGE and Western blot analyses of the phosphorylated form (S139) of H2AX (p-H2AX marker of DSB), H2AX, and actin proteins. Control or etoposide-treated cells were also immunostained with anti–phospho-H2AX antibody. (B) p30 nucleolar delocalization is associated with DSB triggering. HeLa cells transfected with GFP-p30 (green) were treated with etoposide as described in panel A and immunostained with anti–phospho-H2AX antibody showing DSBs (red). (C-D) p30 nucleolar delocalization is specifically induced by DNA-damage agents. HeLa cells transfected with GFP-p30 or GFP-nucleolin (control) were either treated with bleomycin (which induces irreversible DNA breaks) or γ-irradiated (5 Gy). (E) The C-terminal end of the p30 protein includes the response motif for p30 nucleolar delocalization. HeLa cells were transfected with a series of C-terminal–deleted mutants of p30 all known to localize in the nucleolus (GFP-p30delC6, GFP-p30delC5, GFP-p30delC4, and GFP-p30delC3), and treated with etoposide as described in panel A. Only cells expressing wild-type p30 responded to the etoposide treatment. (F) Representation of p30 C-terminal–deleted mutants. (G) Thr232 is required for p30 delocalization in response to DSB. HeLa cells were transfected with GFP-p30 or GFP-p30T232A and treated with etoposide in the presence or the absence of PD98059, a specific MAPK inhibitor. (H) HeLa cells were transfected with GFP-p30 or the GFP-p30T232A mutant, γ-irradiated, and stained with p-H2AX antibody. (I) The threonine at position 232 is phosphorylated. 293T cells were transfected with pMH-, p30-HA-, or p30-T232A-HA–expressing vectors. After transfection (48 hours), the cells were irradiated (5 Gy), and 2 hours later they were lysed and cell extracts were immunoprecipitated with anti-HA antibody. Western blot analysis was then performed with an anti–phospho-threonine-proline–specific antibody.

p30 protein relocalizes on DNA-damage–induced DSBs. (A) Etoposide induces DNA DSBs. HeLa cells were left untreated or treated with etoposide for 24 hours, followed by SDS-PAGE and Western blot analyses of the phosphorylated form (S139) of H2AX (p-H2AX marker of DSB), H2AX, and actin proteins. Control or etoposide-treated cells were also immunostained with anti–phospho-H2AX antibody. (B) p30 nucleolar delocalization is associated with DSB triggering. HeLa cells transfected with GFP-p30 (green) were treated with etoposide as described in panel A and immunostained with anti–phospho-H2AX antibody showing DSBs (red). (C-D) p30 nucleolar delocalization is specifically induced by DNA-damage agents. HeLa cells transfected with GFP-p30 or GFP-nucleolin (control) were either treated with bleomycin (which induces irreversible DNA breaks) or γ-irradiated (5 Gy). (E) The C-terminal end of the p30 protein includes the response motif for p30 nucleolar delocalization. HeLa cells were transfected with a series of C-terminal–deleted mutants of p30 all known to localize in the nucleolus (GFP-p30delC6, GFP-p30delC5, GFP-p30delC4, and GFP-p30delC3), and treated with etoposide as described in panel A. Only cells expressing wild-type p30 responded to the etoposide treatment. (F) Representation of p30 C-terminal–deleted mutants. (G) Thr232 is required for p30 delocalization in response to DSB. HeLa cells were transfected with GFP-p30 or GFP-p30T232A and treated with etoposide in the presence or the absence of PD98059, a specific MAPK inhibitor. (H) HeLa cells were transfected with GFP-p30 or the GFP-p30T232A mutant, γ-irradiated, and stained with p-H2AX antibody. (I) The threonine at position 232 is phosphorylated. 293T cells were transfected with pMH-, p30-HA-, or p30-T232A-HA–expressing vectors. After transfection (48 hours), the cells were irradiated (5 Gy), and 2 hours later they were lysed and cell extracts were immunoprecipitated with anti-HA antibody. Western blot analysis was then performed with an anti–phospho-threonine-proline–specific antibody.

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