Figure 2. IR-induced p53 phosphorylation at Ser15 plays a crucial role in IR-inducible miR-34a expression via the p38 MAPK pathway. (A) Whole cellular lysate was prepared from IR-exposed HMEC cells at the indicated time-points post-irradiation; western blot analysis was performed using antibodies to p-p53, p53, p21 and actin. (B) Total RNA was isolated from HMEC cells exposed to IR at the indicated time-points; the levels of hsa-miR-34a were examined by real-time RT-PCR, as described in “Material and Methods.” (C) Whole cellular lysate was prepared as described in (A); western blot analysis was performed using antibodies to p-ERK1/2 or ERK1/2, p-p38 or p38, p-RSK2 or RSK2, p-MSK1 or MSK1, and actin. (D) Whole cellular lysate was prepared from HMEC cells exposed to IR in combination with different concentrations of SB239063 96 h post-irradiation; western blot analysis was performed using antibodies to p-p53, p53, p-p38, p38 and actin. (E) Total RNA was isolated from HMEC cells exposed to IR (upper penal, 30 kVp/0.1 Gy; lower penal, 80 kVp/2.5 Gy) in combination with different concentrations of SB239063 96 h post-irradiation; quantitative real-time RT-PCR was performed using hsa-miR-34a primers. (F) HEK293 cells grown to 90% confluency were cotransfected with either wild-type miR-34a promoter reporter or mutant miR-34a promoter reporter and either pEGFP-C1/WT-p53 or pEGFP-C1/MT-p53; 24 h after transfection, the cell lysate was subjected to luciferase assay, as described in “Materials and Methods.” (G) HMEC cells grown to 90% confluency were transfected with either pEGFP-C1/WT-p53 or pEGFP-C1/MT-p53; 96 h after transfection, real-time ChIP-PCR was performed, as described in “Materials and Methods.” The asterisk indicates p < 0.05.