Response: Location of BRCA1 in Human Breast and Ovarian Cancer Cells

1. Y. Chen et a/. , Science 270, 789 (1995). 2. Y. Miki et a/. , ibid. 266, 66 (1994). We obtained the cDNA for BRCAl by probing a 293-cell cDNA library. Two partially overlapping fragments were reassembled to produce full-length BRCAl cDNA, which was then sequenced and found to contain no mutations. Antibodies were raised in mice against a BRCAl -GST fusion protein that contained residues 1 to 304 of BRCAl (amino terminal segment, MS series of mAbs) or residues 1313 to 1863 of BRCA1 (carboxyl-terminal segment, AP series of mAbs). The SG series of mAbs was raised against the BRCAl carboxyl-terminal peptide CQELDTYLIPQIPHSHY. Monoclonal antibody fusions were screened by immunoprecipitation of 35S-methionine-labeled, in vitro-translated BRCA1, followed by 6% SDS-PAGE and autoradiography. Positive wells were recloned and rescreened twice, the second subcloning being at limiting dilution, which allowed single-cell clones to be picked. 3. R. Eckner et al., Genes Dev. 8, 869 (1994). 4. Alcoholic formalin is composed of 10% buffered formalin (Anatech Ltd, Battle Creek, Ml) diluted into 70% ethanol. Immunoperoxidase staining was performed using a Ventana Automated Immunostainer (Ventana Medical Systems, Tucson, AZ). Monoclonal antibody SG11 was used at 1:10 dilution; mAb MS13 was used without dilution. Where microwave heating was used for antigen retrieval, sections were heated in the microwave in citrate buffer pH 6 for 10 minutes, with buffer replacement in between the heating periods. 3-3' diaminobenzadine was used as the chromogen, and the sample was lightly counterstained with methyl green. 5. Cells were lysed in RIPA buffer (50 mM tris, pH 8.0, 150 mM sodium chloride, 0.1% SDS, 0.1% sodium deoxycholate, 1 % NP 40) in the presence of phosphatase inhibitors (50 mM sodium fluoride, 1 mM sodium orthovanadate) and protease inhibitors [aprotinin (20 pg/ml), leupeptin (10 ,ug/mI), phenylmethylsulphonylfluoride (100 pg/ml)], at 40C. After separation by 6% SDS-PAGE, proteins were transferred to PVDF (Immobilon P, Millipore, Bedford, MA) by electrophoresis in Towbin buffer (25 mM tris, 192 mM glycine, pH 8.3, 0.01% SDS, 0% methanol). The membrane was blocked in either 4% bovine serum albumin (BSA) or 5% nonfat milk in tris-buffered saline (TBS) (0.9% sodium chloride, 20 mM tris, pH 7.4) supplemented with 0.1% sodium azide and 0.05% Tween 20, for 1 hour at room temperature. The primary antibody was mAb MS1 10 (2) at 1 :10 dilution of tissue-culture supernatant in TBS-Tween 20 0.05% (TBS-T), with 2% BSA or nonfat milk and 0.1% sodium azide. Washes were in TBS-T. The secondary antibody was affinity-purified goat antibody to mIgGperoxidase (Boehringer-Mannheim, Indianapolis, IN). The signal was developed using ECL (Amersham, Arlington Heights, IL). 6. W. -H. Lee et al., Nature 329, 642 (1987). Cells were not labeled prior to fractionation. The cytosolic fraction was concentrated using a regenerated cellulose 10,000 NMWL filter unit (ultrafree-MC, Millipore) and equilibrated with lysis buffer. The protein concentration of each fraction was measured with reference to a standard curve, using a detergent-compatible protein assay (Bio-Rad, Hercules, CA). Conditions for the protein immunoblot are given above (5). 7. We thank J. Zhou and J. Prioleau for technical assistance. R.S. was supported by a grant from the Dana-Farber Women's Cancer Program. Mouse mAb GG22 (Fig. 1) was a gift from D. Ginsberg; mouse mAb RW128 (Fig. 3) was a gift from R. Eckner; and rabbit polyclonal antiserum to GDI-1 (Fig. 3) was a gift from P. E. Bickel. This work was also funded by grants from the U.S. National Institutes of Health.