Fig. 6.

γH2AX coprecipitates with PP1α and Cavin3 following UV stress. a A431 cells were treated with tautomycin (0.5 μM) for 3 h prior to UV irradiation. After a 4 h chase, cells extracts were western blotted for γH2AX and H2AX levels. β-Actin was blotted as the loading control. b γH2AX expression levels were quantified from three independent experiments where the data is presented as mean ± SD. Unt (−TTM): 100.0 ± 0.0%; Unt (+TTM): 155.1 ± 10.7%; UV (−TTM): 108.8 ± 31.5%; UV (+TTM): 215.2 ± 30.1%) where *p = 0.0466 was calculated using an ordinary one-way ANOVA. c, d GFP Trap assays of A431 cells transfected with GFP-PP1α or GFP (c), GFP-Cavin3 or GFP (d) followed by western bloting with an anti-γH2AX antibodies. Transfection efficiency was confirmed with GFP antibodies. α-Tubulin was used as the loading control. e, g PLA of the interaction of endogenous PP1α (e) or Cavin3 (g) with γH2AX upon UV radiation (shown as red dots). PLA signals alone were inverted to gray scale. Scale bar, 10 μm. Mean ± SD from three independent experiments are presented on the images. f, h The number and ratio of the PLA signals for PP1α (f) or Cavin3 (h) with γH2AX in the cytoplasm (gray) and nucleus (purple) were calculated and presented in the histograms and tables as mean ± SD from three independent experiments. i Phosphatase activity of PP1α in MCF-7 cells with or without mScarlet-PP1α (mS- PP1α) or/and pCB6-Cavin3 transfection. Relative phosphatase activity of control (%) (Ctrl: 100.0 ± 0.0%; mS-PP1: 123.4 ± 8.3%; Cavin3: 77.6 ± 10.0%; mS-PP1+Cavin3: 93.5 ± 7.4%) is presented as the mean ± SD from three independent experiments (Ctrl vs mS-PP1: *p = 0.0209, Ctrl vs Cavin3: *p = 0.0260, mS-PP1 vs mS-PP1+Cavin3: **p = 0.0053) using an ordinary one-way ANOVA