FIG. 4.
Knockdown of PP6c or PP6R1 induces sustained phosphorylation of γ-H2AX in irradiated cells. (A) HeLa cells either were left untransfected (lanes 1 to 5) or were transfected with a siRNA to either PP6c (lanes 6 to 10) or a scrambled RNA control (lanes 11 to 15). Where indicated (+), cells were irradiated with 10 Gy and were collected at the times indicated after IR. The minus sign (−) above lanes 1, 6, and 11 indicates unirradiated cells. The soluble fraction of detergent extracts was analyzed by SDS-PAGE and immunoblotted with antibodies to PP6c or Ku80 (Ku80sol) as indicated. The pellet remaining after detergent lysis was resuspended in SDS as described in Materials and Methods and was analyzed for γ-H2AX phosphorylation or Ku80 (Ku80pell) as indicated. Extracts were also probed for the expression of other protein phosphatase catalytic and regulatory subunits (shown in Fig. S4 in the supplemental material). (B) The signal intensity of γ-H2AX was normalized to that of Ku80, and the relative phosphorylation of H2AX in arbitrary units is plotted. Gray bars represent extracts from cells transfected with a siRNA to PP6c; black bars represent extracts from cells transfected with a scrambled control siRNA. (C) Cells were transfected with a siRNA to PP6R1, and extracts were generated and analyzed exactly as for panel A. Further controls are shown in Fig. S6 in the supplemental material. (D) Quantitation of γ-H2AX phosphorylation as in panel B. Gray bars represent extracts from cells transfected with a siRNA to PP6R1; black bars represent extracts from cells transfected with a scrambled control siRNA.