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RAD51AP2 and RAD51 proteins interact in human cells, and the interaction is enhanced by DNA damage

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posted on 2011-12-31, 02:53 authored by Oleg V. Kovalenko, Claudia Wiese, David Schild

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Taken from "RAD51AP2, a novel vertebrate- and meiotic-specific protein, shares a conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51"

Nucleic Acids Research 2006;34(18):5081-5092.

Published online 20 Sep 2006

PMCID:PMC1636435.

Published by Oxford University Press 2006

() Human HEK293 cells were transiently transfected with plasmids encoding the following T7 epitope-tagged proteins: Vmw65 protein of herpesvirus (lane 1); 293amino acid RAD51AP2 polypeptide (lane 2); RAD51AP2 protein deleted for its last 57 residues (lane 3). Immunoprecipitation with anti-T7 antibody followed by protein A-agarose beads was carried out, and fractions of cell lysates before IP, supernatant of IP, and IP pellets were analyzed by Western blotting using anti-T7 (top panel) or anti-RAD51 antibody (lower panel). Secondary antibodies were conjugated to alkaline phosphatase, which was detected using chromogenic substrates. () Transfection with plasmid encoding the 293 amino acid RAD51AP2 polypeptide was performed as in panel A, and 0.5 μg/ml MMC, 100 μg/ml MMS or 10 μg/ml cycloheximide (Cyc.) was added to individual cultures 3 h before harvesting cells for IP. IP and Western blotting were done as in panel A, except that chemiluminescent substrates were used to detect peroxidase conjugated to secondary antibodies. To facilitate the relative comparison of RAD51 signals from IP pellets (shown in panel C), the amount of these fractions loaded on the gel was adjusted in an effort to get similar intensities of the T7-tagged RAD51AP2 signal in each lane. The residual differences were normalized to the control (untreated cells) for comparison of the amounts of RAD51 that coprecipitated with RAD51AP2. () Quantitation of RAD51-RAD51AP2 coimmunoprecipitation from panel B. The signals from precipitated RAD51 in IP pellet fractions were measured using a densitometer and then normalized for the signal from anti-T7 analysis of the same fractions. The relative increase of RAD51 signal from treated cells as compared to the signal observed from untreated cells (artificially set at 1.0 in left lane) is shown.

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