FIGURE 3.
IR-induced degradation of p21Cip1 is independent of ATM. A, Western blot of whole cell lysates from HEK293 cells pretreated for 1 h with DMSO or 10 μm KU55933 prior to 10 Gy IR. Phospho-specific antibodies to ATM or the ATM target Chk2 were used to verify the efficacy of the inhibitor. B, Western blot of whole cell lysates from immortalized A-T fibroblasts or reconstituted A-T fibroblasts pretreated with 25 μg/ml cycloheximide for 1 h prior to 10 Gy IR. C, Western blot of whole cell lysate from HEK293 cells pretreated with 25 μg/ml CHX and DMSO or 100 μm wortmannin for 1 h prior to 10 Gy IR. Phospho-specific antibodies to ATM or ATR target sites were used to verify the efficacy of the inhibitor. D, A-T cells were transfected with siRNAs to ATR or DNA-PK, either individually or in combination. 60 h post-transfection, cells were pretreated with 25 μg/ml CHX for 1 h prior to irradiation with 10 Gy IR. Cells were collected 1 h after IR, and equal amounts of whole cell lysate were analyzed by Western blot.