Fig. 2.
MCM3 is phosphorylated by ATM. (A) GST-MCM3 was incubated with WT or kinase-dead (KD) Flag-ATM or Flag-ATR in the presence of [γ]-[32]ATP. The kinase reaction was separated by SDS/PAGE. An autoradiogram and picture of the Coomassie blue-stained MCM3 protein is shown, as well as an anti-Flag Western blot showing the levels of ATM and ATR protein. (B) WT GST-MCM3 or S535A-mutated GST-MCM3 was subjected to kinase reactions with ATM or ATR. An autoradiogram of the 32P incorporation into the MCM3 proteins is shown, as well as a picture of the Coomassie-stained gel. (C) AT22IJE-T (homozygous ATM mutant cells, A-T) or AT22IJE-T cells containing a vector encoding ATM were left untreated or exposed to 10 Gy of IR and harvested after 1 or 6 h. Protein from these cells was fractionated by SDS/PAGE and immunoblotted with either the SQ2 or MCM3 antibody. (D–F) Lysates were prepared from either ATR+/+ or ATRflox/- cells after infection with adenovirus encoding either Cre or GFP and treatment with HU (D), UV light (E), or IR (F) and harvested after the indicated number of hours. Proteins were fractionated by SDS/PAGE, blotted, and probed with either the SQ2 or MCM3 antibody. (G) U2OS cells were transfected with siRNA targeting ATM and/or ATR, exposed to UV light (50 J/m2), incubated for 2 h, and then harvested. Protein lysates were fractionated by SDS/PAGE, blotted, and probed with antibodies as indicated.