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. 2016 Oct 7;14(6):804–813. doi: 10.1080/15476286.2016.1243647

Figure 3.

Figure 3.

Phosphorylated WRAP53β accumulates at sites of DNA damage. (A) Schematic illustration of the LacO-LacI-FokI system in U2OS cells. (B) Following a 5 h induction of the mCherry-LacI-FokI fusion protein, the U2OS-FokI cells were fixed and immunostained for γH2AX to confirm the generation of double-strand breaks. (C) Following a 5 h induction of double-strand breaks by the mCherry-LacI-FokI fusion protein, the U2OS-FokI cells were fixed and immunostained with the antibodies indicated. (D) Parental U2OS cells were micro-irradiated, fixed 15 min later and immunostained for γH2AX and pWRAP53βS64. (E) Parental U2OS cells were either left untreated or irradiated with 6 Gy (1 hour recovery), pre-extracted using CSK buffer, fixed and immunostained for γH2AX and endogenous WRAP53β using the 1F12 antibody. (F) U2OS cells stably expressing Flag-WRAP53β were either left untreated or exposed to 6 Gy IR or 30 J/m2 UV, allowed to recover for periods of time indicated, then immunostained for pWRAP53βS64, γH2AX or RPA2. (G) U2OS cells were transiently transfected with the plasmids indicated for 24 h, fixed and stained for the Cajal body marker protein coilin. (H) U2OS cells were irradiated with 6 Gy (1 hour recovery), pre-extracted using CSK buffer, fixed and immunostained for WRAP53β using indicated antibodies and for coilin. In all immunofluorescent stainings, nuclei were stained with DAPI.