Figure 1.

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.