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Sheep anti Human MDC1 antibody (AHP799) used for the visualization of MDC1 expression in U2OS osteosarcoma cells following irradiation induced DNA damage by immunofluorescence.
PP4 dephosphorylates γH2AX at the sites of DNA damage. (A,B) U2OS cells transfected with siRNAs against PP4C, PP2ACα/β or a control sequence were irradiated with 1.5 Gy and processed for γH2AX (A,B) and MDC1 (B) immunofluorescence and DAPI counterstaining at the indicated time points. Images were taken with a × 40 objective (A) or with a × 63 objective (B). Scale bar, 23 μm (A) and 16 μm (B). (C–F) Quantification of cells with >5 γH2AX (C), >10 γH2AX (D,F) and >5 MDC1 (E) foci. At least 300 cells were analysed per experiment. Cells were treated with KU55933 and KU57788 12 min post-IR (F). Error bars represent standard error of mean of independent duplicated experiments (C) or standard deviation of three independent experiments (D–F). A × 63 oil immersion objective (C,E) or a × 40 water immersion objective (D,F) was used. (G) U2OS cells transfected with siRNAs against PP4C or a control sequence were collected at the indicated time points immediately before (no IR), or 1 or 6 h after irradiation (10 Gy), then fractionated as described in Méndez & Stillman (2000) into whole-cell lysates (WCLs), nuclear soluble (S3) or chromatin (P3) fractions, and analysed by immunoblotting using the indicated antibodies. DAPI, 4,6-diamidino-2-phenylindole; H3, histone H3; IR, irradiation; MDC1, mediator of DNA damage checkpoint 1; siRNA, small interfering RNA.
From: Nakada S, Chen GI, Gingras AC, Durocher D.
PP4 is a gamma H2AX phosphatase required for recovery from the DNA damage checkpoint.
EMBO Rep. 2008 Oct;9(10):1019-26.