Figure 2
DNA-PKcs activation is required for UVB-induced Akt Ser-473 phosphorylation. UVB radiation induced DNA-PKcs phosphorylation in primary skin keratinocytes (A), DNA-PKcs phosphorylation (Thr 2647) was quantified. UVB(UV, 30 mJ/cm2, 1 hr)-induced Akt Ser-473 and Thr-308 phosphorylation in primary skin keratinocytes transfected with scramble or two distinct DNA-PKcs siRNAs (“-1” was from Santa Cruz, “-2” was from Dharmacon) (100 nM each). Expressions of DNA-PKcs, SIN1 and p- and t- Akt/Erk were also tested (B), Akt phosphorylation was quantified. HaCaT cells transfected with scramble or DNA-PKcs siRNA-2 (100 nM each) were left untreated or irradiated with UVB (UV, 30 mJ/cm2) for 1 hr, followed by immuno-blot detecting Akt (p- and regular), DNA-PKcs, mTOR and β-actin (C), Akt phosphorylation was quantified. Effect of NU7026 (10 μM, 1 hr pretreatment) on UVB (UV, 30 mJ/cm2)-induced Akt, S6 and Erk1/2 phosphorylation was shown, regular Akt, Erk and S6 were also shown (D), Akt and S6 phosphorylation was quantified. Primary keratinocytes, transfected with vector, dominant negative kinase dead DNA-PKcs (T2609A, DN) or WT-DNA-PKcs (1.0 μg/ml each), irradiated with UVB (UV, 30 mJ/cm2) for 1 hr, DNA-PKcs, Akt (p- and regular), Erk (p- and regular) were tested (E), Akt phosphorylation was quantified. WT and DNA-PKcs KO MEFs were irradiated with UVB (UV, 30 mJ/cm2) for 1 hr, followed by immunoblotting detecting DNA-PKcs, Akt (p- and regular) and mTOR (F), Akt phosphorylation was quantified. WT and DNA-PKcs KO MEFs were treated with insulin (200 nM) for 10 min, p- and t- Akt/Erk1/2 as well as DNA-PKcs expression were tested (G). p < 0.05 vs. 0 or Ctrl (A and B). **p < 0.05 vs. control group (C-F). #p > 0.05 vs. control group (C-F).