Figure 3
DNA-PKcs-SIN1 cytosol association mediates UVB-induced Akt Ser-473 phosphorylation. Effect of NU7026 (10 μM, 1 hr pretreatment) on UVB (UV, 30 mJ/cm2)-induced DNA-PKcs/SIN1/mTOR association was tested by Co-IP (A and B), SIN1-DNA-PKcs association was quantified. The expression of DNA-PKcs/mTOR/SIN1 as well as S6 phosphorylation in whole cell lysates were also shown (C, input). WT and SIN1 KO (−/−) MEFs were irradiated with UVB (UV, 30 mJ/cm2) and cultured for 1 hr, followed by immunoblotting detecting p-Akt, regular Akt and GSK3β (D). Primary skin keratinocytes, transfected with dominant negative (DN) DNA-PKcs, scramble siRNA or DNA-PKcs siRNA (“-2”), were irradiated with UVB (UV, 30 mJ/cm2) and cultured for 15 min, the association between SIN1/DNA-PKcs was tested by Co-IP (E), SIN1 bound DNA-PKcs was quantified. Cytosol and nuclear DNA-PKcs, Akt and SIN1 in UVB (UV, 30 mJ/cm2) irradiated keratinocytes (F), cytosol and nuclear DNA-PKcs was quantified. *p < 0.05 (A), *p < 0.05 vs. 0′ or Ctrl (E and F).