Characterization of BRCA2 protein. (A) Diagram showing the relative positions of peptides (N19 and C15) and GST-fusion proteins (GSTB1, GSTB2, and GSTB3) used to generate antibodies against BRCA2. Among antibodies generated against GST-fusion proteins, we selected a mAb generated against GSTB2 for use in this study. (B) Lysates (50 μg of total protein) of 293T cells transfected with pGFPB2 (lanes 1, 3, 5, 7, and 9) or control vector pEGFPC1 (lanes 2, 4, 6, 8, and 10) were subjected to 4% SDS/PAGE, and immunoblotted with the indicated antibodies with (lanes 3, 4, 7, and 8) or without (lanes 1, 2, 5, 6, 9, and 10) homologous peptide competition. The GFP tag at the N terminus of BRCA2 may interfere with recognition of BRCA2 by anti-N19, which is specific for the N terminus of BRCA2, based on the relatively stronger signal for GFP-BRCA2 using anti-C15. (C) Cell lysates (50 μg of total protein; lanes 1–4), mAb B2 immunoprecipitates (from 1 mg of total protein; lanes 5–8), and DNA-dependent protein kinase immunoprecipitates (1 mg of total protein; lane 9) were resolved on the same 4% SDS/PAGE gel, and immunoblotted with anti-C15 or anti-DNA-protein kinase. Cells included 293T transfected with pEGFPC1 (lanes 1 and 5) or pGFPB2 (lanes 2 and 6), MCF7 (lanes 3, 7, and 9), and CAPAN-1 (lanes 4 and 8). (D) mAb B2 immunprecipitates from 2 mg of total protein of CAPAN-1 (lane 1) and MCF7 (lane 2) were resolved by 5% SDS/PAGE and immunoblotted with anti-N19. (E) MCF7 (lane 1) and CAPAN-1 (lane 2) cell lysates were labeled with [³²P]phosphoric acid, immunoprecipitated with mAb B2, resolved on a 4% SDS/PAGE, and exposed to Kodak X-Omat AR film. (F) BRCA2 or RB immunoprecipitates of MCF7 cells were untreated (lanes 1 and 3) or treated (lanes 2 and 4) with lambda-phosphatase, separated by 4% (BRCA2) or 8% (RB) SDS/PAGE, and immunoblotted with anti-C15 or anti-RB.

Subcellular localization of exogenously expressed BRCA2. Representative images of live 293T cells transfected with pGFPB2. (A) Image showing the green signal of GFP-BRCA2 in transfected 293T cells. (B) Image showing the nuclei of cells in the same field labeled with Hoechst. Hoechst labeling is indicated as red color instead of its authentic blue color. (C) Overlay of the images in A and B. (Bar = 10 μm.)

In vivo interactions of BRCA2 with RAD51. (A) Cell lysates (50 μg of total protein; lanes 1 and 2) and mAb B2 (lanes 3 and 4) or anti-IgG (lane 5) immunoprecipitates (from 2.5 mg of total protein) were resolved by 10% SDS/PAGE, and immunoblotted with anti-RAD51. (B) Cell lysates (50 μg of total protein for CAPAN-1, and 25 μg of total protein for MCF7; lanes 1 and 2) and mAb B2 immunoprecipitates (from 4 mg of total protein for CAPAN-1, and 2 mg of total protein for MCF7; lanes 3 and 4) were resolved by 10% SDS/PAGE and immunoblotted with anti-RAD51. (C) 293T cells transfected with pHA-RAD51 or the control HA vector were lysed and immunoprecipitated with anti-HA, mAb B2, or anti-IgG. Lysates (lanes 1 and 2) and immunoprecipitates (lanes 3–5) were separated by 4% SDS/PAGE and immunoblotted with anti-C15, or 10% SDS/PAGE and immunoblotted with anti-HA. Anti-IgG immunoprecipitates from HA-RAD51 transfected cells were used as a specificity control (lane 5).

BRCA2 forms a complex with p53 in vivo. (A) Cell lysates (40 μg of total protein; lanes 1–3) and p53 immunoprecipitates (4 mg of total protein; lanes 4–6) were resolved by 6% SDS/PAGE and immunoblotted with anti-C15. (B) Cell lysates (lanes 1–3) were separated by 10% SDS/PAGE and immunoblotted with anti-p53 PAb421. Anti-C15 immunoprecipitates from [³⁵S]-methionine-labeled cells were eluted and reprecipitated with anti-p53 DO-1. The double immunoprecipitates were resolved by 10% SDS/PAGE and subjected to fluorography (lanes 4–6).

RAD51 enhances BRCA2 inhibition of p53’s transcriptional activity. MCF7 cells were cotransfected with the indicated plasmids and PGluc. Forty-eight hours after transfection, luciferase activity was measured as described in Materials and Methods.