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. 2018 Oct 16;50(10):136. doi: 10.1038/s12276-018-0169-z

Fig. 1. BRCA1 interacts with cyclin B1.

Fig. 1

a MCF7 cells were transfected with a construct encoding His-tagged BRCA-Δ11 and irradiated to induce DNA damage. Extracts of transfected MCF7 cells were prepared and subjected to Ni-NTA affinity chromatography, and the resulting eluates were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining. The arrow indicates cyclin B1 further determined by matrix -assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. b Lysates of 293T cells transfected with Myc-BRCA-Δ11 and/or FLAG-cyclin B1 were immunoprecipitated with anti-FLAG antibody-conjugated agarose. The resulting precipitates as well as the original lysates (Input) were analyzed by immunoblotting with the indicated antibodies. c Lysates of irradiated MCF7 cells were immunoprecipitated with control or anti-BRCA1 IgG, followed by Western blot analyses using the indicated antibodies. d The 293T cells transfected with Myc-BRCA1-Δ11 and FLAG-cyclin B1 were irradiated (10 Gy) and immunoprecipitated with anti-FLAG antibody-conjugated agarose, after which interactions were assessed by Western blot analysis of immunoprecipitates using the indicated antibodies. e Lysates of MCF7 cells in the absence or presence of irradiation were immunoprecipitated with an antibody against BRCA1, followed by Western blot analyses using the indicated antibodies. f Myc fusion constructs of BRCA1-Δ11 and deletion mutants. RING Really Interesting New Gene finger domain, BRCT BRCA1 C-terminal domain. g FLAG fusion constructs of cyclin B1 and deletion mutants. D-box destruction box domain CRS/NES, cytoplasmic retention signal/nuclear export signal domain. h Lysates of 293T cells transfected with FLAG-cyclin B1, Myc-BRCA1-Δ11, Myc-BRCA1 N-terminal fragment, and/or Myc-BRCA1 C-terminal fragment constructs were immunoprecipitated with anti-FLAG antibody-conjugated agarose and analyzed by immunoblot analysis with the indicated antibodies. i Lysates of 293T cells transfected with Myc-BRCA1-Δ11 or several forms of FLAG-cyclin B1 constructs were immunoprecipitated with anti-FLAG antibody-conjugated agarose, and immunoprecipitates were analyzed by immunoblotting using the indicated antibodies. Input represents 5% of the protein extract prior to immunoprecipitation