Fig 1: Analysis of DT40 and human cell survival after MMS treatment, as a function of BRCA1 and pol ß expression and combined inactivation of BRCA1 and pol ß.Experiments were conducted as described under “Materials and Methods”. A. DT40 BRCA1+/+ (wild-type), BRCA1-/-, Pol ß-/- and BRCA1-/-/Pol ß-/- double knockout cells were treated with MMS and survival was measured by colony formation. B. Human BRCA1-expressing (+) and negative (-) cells were treated with MMS in the presence or absence of the pol ß inhibitor PA (300 µM for 24 h). Results with error bars represent the mean ± SEM of at least 3 independent experiments.
Fig 2: Co-immunoprecipitation analysis for human BRCA1 and pol ß interaction.Experiments were conducted as described under “Materials and Methods”. Typical results obtained in at least three experiments are shown. Immunoprecipitated proteins were detected by SDS-PAGE and immunoblotting (IB). A. Cell extracts, as indicated, were immunoprecipitated with anti-pol ß antibody (lanes 2–5) or non-immune IgG (lane 1). Lanes 2–3 and 4–5 are duplicates. Lane 6 corresponds to immunoblotting of the BRCA1-complemented extract, used here as a source of markers for BRCA1 and pol ß. B, Cell extracts as indicated were immunoprecipitated with anti-BRCA1 antibody (lanes 2–5) or non-immune IgG (lane 1). Lanes 2–3 and 4–5 are duplicates, and lane 6 was as described in panel A. C. Purified human BRCA1 and pol ß proteins were mixed and immunoprecipitated with anti-pol ß antibody (lane 1) or non-immune IgG (lane 3). Pol ß was omitted in lane 2, and lane 4 corresponds to the purified proteins used as markers. D. The same purified protein mixture as in panel C, except that immunoprecipitation was with anti-BRCA1 antibody (lane 1) or non-immune IgG (lane 3). BRCA1 was omitted in lane 2.
Fig 3: Characterization of MMS-induced DNA damage in human and DT40 BRCA1 cell lines.Experiments were conducted as described under “Materials and Methods”. A. Human BRCA1 positive (+) and negative (-) cells were treated for 1 h with MMS as indicated, and survival was measured. B. Chicken DT40 BRCA1+/+ (wild-type) and BRCA1-/- cells were treated continuously with MMS, and survival was measured by clonogenic assay. Results with error bars represent the mean ± SE of at least 3 independent experiments; other data points represent the mean of two experiments. C. DT40 BRCA1+/+ and BRCA1-/- cells were treated with 20 mM MMS for 20 min on ice and then subjected to the alkaline comet assay for measurement of strand breaks after 0 and 60 min repair. 100 cells were scored per slide, 2 slides per cell type and time, and migrated DNA was measured as the Mean % Tail DNA ± SD of 2 replicate slides. Mean % Tail DNA was normalized to the untreated Mean % Tail DNA in the respective cell line. The difference between cell types in level of DNA damage was significant (p = 0.003).
Fig 4: Immunofluorescence imaging of pol ß and BRCA1 in human cells using anti-pol ß and anti-BRCA1 antibodies.Experiments were conducted as described under “Materials and Methods,” and typical results are shown. A. BRCA1 positive and B. BRCA1 negative cells were irradiated in stripes and then allowed to repair for the indicated times before assessment for pol ß and BRCA1 recruitment, as indicated. C. Summary of intensities of the pol ß and BRCA1 signals as a function of time after irradiation of the BRCA1-expressing cells. Quantification was of at least 18 cells for each protein with error bars representing mean ± SD. D. Summary of intensities of the pol ß signals, as a function of time after irradiation, in the BRCA1 positive cells (+, solid bars) and negative cells (-, open bars). Values plotted represent mean ± SD corresponding to 4 cells at each point. E. Imaging of ?-H2AX in the BRCA1-expressing (+) cells 15 min after irradiation. Bars represent 10 µm.
Fig 5: Analysis of pol ß KD cells.A. Western blotting analysis of pol ß in a pol ß knockdown (KD) cell line and in a partial revertant. B and C. Immunofluorescence imaging of the recruitment of pol ß and BRCA1 at 1 and 15 min after irradiation of BRCA1+, pol ß KD and pol ß KD revertant cell lines. Bars represent 10 µm.
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