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. 2006 Jan 1;20(1):34–46. doi: 10.1101/gad.1381306

Figure 2.

Figure 2.

Damage-induced BRCA1 interactions with TopBP1 and the M/R/N complex. (A) Silver-stained gel demonstrating purified complexes after single Flag (Flag) and consecutive Flag and HA purification steps (Flag/HA) for eBARD1 (B) and mock (M) transduced HeLa S3 cell lines. The BRCA1 and BARD1 bands are denoted in bold black type. Selected interacting partners that had been reported previously are indicated in nonbold black type. Novel interacting proteins are indicated with a red asterisk (*). (B) Complexes were purified 3 h after 0 (–) or 10 Gy (+) by serial Flag and HA-immunoaffinity chromatography/elution (Flag/HA). BRCA1 bands from undamaged cells and post-IR phosphorylated BRCA1 (BRCA1-P) are indicated. Damage-specific bands are indicated by red asterisks (*). A new band for NBS1 was not detected due to its comigration with BARD1. (C) Immunoblot detection of IR-dependent TopBP1 and NBS1 association with Flag/HA-purified BARD1-containing complexes. Damage-induced binding proteins are indicated in red. (D) Flag/HA-purified eBARD1 complexes were isolated from HeLa S3 cells following mock treatment, IR, or a thymidine block. Damage-activated binding events are indicated in red. (E) IF for H2AX-P and TopBP1 in microirradiated HCC1937 cells reconstituted with vector (top panels) or wild-type BRCA1 (bottom panels).