Figure 3.

BRCA1 physically interacts with Nrf2 and affects Keap1-mediated Nrf2 ubiquitination. (A) 293FT cells were transfected with Myc-BRCA1 and GFP-NRF2 constructs and then left untreated (Con) or treated with BSO. BRCA1 was immunoprecipitated with anti-Myc Ab. The blot was probed with anti-Myc and anti-GFP Abs. “−” indicates untreated EV control. Vinculin was used as a loading control. (B) COMMA-1D cells were left untreated (Con) or treated with BSO and processed for immunoprecipitation (IP) with anti-Nrf2 (H-300) or anti-Brca1 (C20) Abs to detect the endogenous Brca1 and Nrf2 proteins. (C) COMMA-1D cells were treated as in B and subjected to immunoprecipitation with anti–mouse Brca1 Ab to detect endogenous Brca1 and Nrf2 complex. Nrf2 was detected with an affinity-purified Ab as described in Materials and methods. (B and C) IgG served as an isotype control. (D) 293FT cells were transfected with constructs expressing HA-ubiquitin, His-KEAP1, GFP-NRF2, and/or Myc-BRCA1 (as indicated). Immunoprecipitation was performed with anti-GFP Ab followed by Western blot to detect ubiquitinated NRF2 (HA, GFP) and His-KEAP1. (E) 293FT cells were transfected with Myc-BRCA1, His-KEAP1, HA–WT NRF2 (WT), HA–⁷⁹EGTE NRF2 mutant (⁷⁹EGTE), and HA–²⁹DLG NRF2 mutant (²⁹DLG). Immunoprecipitation was performed with anti-HA Ab, and Western blot was probed with anti-Myc and anti-His Abs. The asterisk indicates an unspecific band.