Figure 4.

RNF4 is recruited to sites of DNA damage in a SIM-dependent manner. (a) Representative micrographs of mock and V5-RNF4 transfected NIH3T3 cells stained with anit-V5 antibody, anti γ-H2AX antibody and DAPI. Cells were untreated or exposed to 5 Gy of IR and fixed after 2 hr. (b) Endogenous RNF4 accumulates at sites of laser-induced DNA damage that are marked by γH2AX. U2-OS cells were micro-irradiated with a multi-photon laser. After 1,4 and 6 min(s) cells were stained with Dapi and immunostained for γH2AX and RNF4. Scale bars, 10 μm. (c) GFP-RNF4 wt/ΔSIM recruitment to DNA damage induced by multi-photon irradiation. RNF4 wild-type, but not RNF4 lacking functional SIMs, accumulates in DNA damage-containing laser tracks. U2-OS cells expressing GFP-RNF4 wt or GFP-RNF4 ΔSIM were subjected to laser micro-irradiation and the accumulation of RNF4 was monitored at the indicated time points after irradiation. The average of two independent experiments −/+ s.e.m. is presented and representative images for the indicated time points are shown. Scale bar, 10 μm. (d) Western blot analysis showing similar levels of expression of GFP-RNF4 and ΔSIMs-mutant in the U2-OS transfected cells