Figure 2.
(A) Expression of ATM and DNA-PK proteins in pMAT1 stably transfected A-T cells (AT1ABR) and pMAT2 stably transfected normal C3ABR cells. Cells were treated with 5 μM CdCl2 for 8 h. Whole-cell lysates (100 μg) were analyzed by SDS-PAGE and immunoblotted with anti-ATM (Ab-3; Oncogene Science) antibodies (top panels). AT1ABR is the parental A-T cell line containing a homozygous 9-bp in-frame deletion and is capable of producing near full-length but nonfunctional protein. Upon induction of the transfected line, a functional full-length recombinant ATM protein is produced. C3ABR is a control lymphoblastoid cell line expressing normal ATM. Induction of the transfected line reduces ATM expression. Membranes were reblotted with anti-DNA-PKcs antibody (Ab-1; Oncogene Science; bottom panels). (B) Phosphorylation of RPA-p34 after exposure of cells to UVC radiation. AT1ABR cells, transfected with a CdCl2-inducible ATM cDNA expression vector pMAT1, and C3ABR cells, transfected with an inducible antisense ATM cDNA expression vector pMAT2, were treated with 5 μM CdCl2 for 8 h (+) or mock-treated (−). After 8 h of CdCl2 treatment, cells were UVC irradiated (30 J/m2) or mock-irradiated. Eight hours after UV treatment, whole-cell lysates were prepared and RPA-p34 was examined by gel electrophoresis, followed by immunoblotting with anti-RPA-p34 (Ab-3; Oncogene Science) antibody. (C) DNA-PKcs (left) and ATM (right) expression in MO59J and MO59K cells detected in whole-cell lysates by immunoblotting. (D) MO59J and MO59K cells were irradiated with 30 J/m2 UV, and whole-cell lysates were prepared at the indicated times after UV irradiation. RPA-p34 hyperphosphorylation was detected by immunoblotting using anti-RPA-p34 (Ab-3; Oncogene Science) antibody. To ensure equal loading and transfer of the protein samples, the gels and the PVDF membranes were stained with Coomassie blue and colloidal gold solution, respectively.