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. 2009 Mar 13;284(11):7284–7293. doi: 10.1074/jbc.M809158200

FIGURE 4.

FIGURE 4.

PTIP foci formation depends on γH2AX·MDC1·RNF8 but not other DNA damage checkpoint or repair proteins. A, PTIP acts downstream of H2AX·MDC1·RNF8 signaling pathway in response to DNA damage. Cells deficient for various DNA damage checkpoint or repair proteins and their respective wild-type counter-parts or reconstituted cells were infected with retrovirus expressing FLAG-tagged PTIP. Cells were then irradiated (10 Gy), and immunostaining experiments were performed using anti-FLAG and anti-γH2AX antibodies 6 h later. B, both the FHA domain and RING domain of RNF8 are required for PTIP IRIF formation. RNF8-/- MEFs stably expressing HA-tagged PTIP were infected with retroviruses expressing SFB-RNF8, SFB-RNF8-ΔFHA, or SFB-RNF8-ΔRING mutants. Immunostaining experiments were performed using anti-γH2AX and anti-HA antibodies. C, UBC13 is required for PTIP localization at or near the sites of DNA breaks. Wild-type or UBC13-/- MEFs were infected with retroviruses expressing SFB-PTIP. Cells were irradiated (10 Gy), fixed, and immunostained with anti-FLAG and pH2AX antibodies.