Fig 1: Radiation induced expression and activation of TRAF4 and complex formation of TRAF4 with NOX2 and NOX4 in normal lung fibroblasts. (A) Expression of TRAF4 in normal lung fibroblasts and NSCLC cells was assessed by Western blotting. (B) Radiation-induced TRAF4 protein expression was detected by Western blotting. Cell lysates were prepared after irradiation (2 Gy, 4 h). (C) Radiation-induced K63-linkage poly-ubiquitination of TRAF4 was measured by an ubiquitination assay. Cells were transfected with mock, Flag-HA-TRAF4 WT, or Flag-HA-TRAF4 dR mutant. Cell lysates were immunoprecipitated with anti-Flag antibody and subsequently analyzed by Western blotting with anti-K63-linkage poly-ubiquitin (Ub) antibody (WCL, whole cell lysates). (D) Radiation-induced mRNA expression of NOX2 and NOX4 was assessed by qRT-PCR. Cells were harvested after irradiation (2 Gy, 3 h). Error bars, ± SEM (n = 3); * p < 0.05 compared with non-irradiated cells. (E) Radiation-induced protein expression of NOX2 and NOX4 was measured by Western blotting. Cell lysates were prepared after irradiation (2 Gy, 4 h). (F) Effects of TRAF4 knockdown on expression of two NOXs and phosphorylation of p47-phox was assessed by Western blotting. (G) To determine NOX complex formation in response to radiation, interactions between TRAF4 and NOX2, NOX4, or phosphorylated p47-phox were investigated by IP assay.