A. BRCA1 negatively regulates the phosphorylation of AKT. Left panel: Lysates were prepared from HCC1937 cells (BRCA1 inactive) that stably expressed BRCA1 (+) or vector only (−) and analyzed by Western blotting (WB) with the indicated antibodies. Right panel: Lysates from Brca1^+/+or Brca1^tr/tr MEFs were analyzed by WB. B. Increased phosphorylation of AKT in BRCA1 knockdown cells. siRNAs corresponding to the genes for the indicated proteins were transferred into MCF-7 breast cancer cells. After 2 days, lysates were prepared and analyzed by WB with the indicated antibodies. C. BRCA1 negatively regulates the AKT kinase activity. Whole-cell lysates prepared from HCC1937 cells expressing BRCA1 (+) or vector only (−) were subjected to immunoprecipitation (IP) with the anti-AKT antibody (AKT kinase kit, Cell Signaling) and analyzed by WB with the indicated antibodies. Insulin is a specific activator of the AKT pathway. D. BRCA1 negatively regulates the AKT pathway. Lysates from HCC1937 cells were analyzed by WB with the indicated antibodies. PDK1 is a kinase of AKT and GSK-3β is a downstream substrate of AKT.

A. Lysates were prepared from HCC1937 cells expressed BRCA1 or vector with or without 50 µM MG132 treatment for two hours, and analyzed by WB with the indicated antibodies. B. HCC1937 cells expressing BRCA1 or empty vector were transfected with a Myc-tagged ubquitin plasmid in the absence or presence of 50 µM MG132 for two hours. Lysates were analyzed by IP and WB with the indicated antibodies. C. 293T cells transfected with constructs expressing Myc-ubiquitin, HA-BRCA1, HA-BRCA1-mutants were treated with 50 µM MG132 for two hours. Lysates were analyzed by IP and WB with the indicated antibodies. ΔC, C-terminal deletion of BRCA1. C61G, inactive E3 ubiquitin ligase of BRCA1. M1775R, point mutation in BRCT domains.

A. BRCA1 interacts with pAKT in cells. Lysates from MCF7 cells were treated with or without λ phosphatase (PPase) or phosphatase inhibitor (10 mM NaF and 50 mM β-glycerophosphate) and then subjected to IP, followed by WB with the indicated antibodies. 20% input was loaded as a control. B. MCF7 cells were prepared at steady state (sts), starvation (sta) and serum stimulation for times indicated. Lysates were subjected to IP with the anti-BRCA1 antibody, followed by WB with the indicated antibodies. C. BRCA1 interacts with pAKT in vitro. Lysates from MCF7 cells were incubated with various GST-BRCA1 fragments immobilized on Sepharose beads were eluted and analyzed by WB with antibody against the phosphorylated or unphosporylated form of AKT (#9272). 20% input was loaded as a control. Equal loading of GST proteins was confirmed by coomassie blue staining. D. BRCA1-BRCT domains interact with pAKT. Lysates were incubated with GST-BRCA1-BRCT wild-type, M1775R and P1749R mutant proteins immobilized on Sepharose beads. The complex was eluted and analyzed by WB with anti-pAKT antibody. Equal loading of GST proteins was confirmed by coomassie blue staining.

Brca1^+/+ and Brca1^tr/tr MEFs were prepared at steady state (sts), starvation and serum stimulation for times indicated. Immunofluorescence staining showed in A and WB analysis in B. Nuclei were stained with DAPI. C. The expression levels of FOXO3a were decreased in nuclear extracts from Brca1^tr/tr MEFs, analyzed by WB (the upper panel). Expressing the AKT1-S473D mutant resulted in the decrease of FOXO3a in Brca1^tr/tr MEFs (the lower panel). Nuclear extracts from Brca1^tr/tr MEFs transfected with indicated plasmids were analyzed by WB. AKT1 siRNA targeted on 3’ UTR region of AKT1 and only decreased expression of endogenous AKT1, but not affect expression of the transfected flag-AKT1. Lanes 3–5 with the AKT antibody showed the transfected flag-AKT1. WT: wild-type of Brca1 plasmid, Mu: Brca1-BRCT mutant (M1775R, point mutation in BRCT domains). D. BRCA1 affects FOXO3a activity via the AKT pathway. mRNA levels of p27^kip1 in Brca1^tr/tr MEFs transfected with indicated plasmids or siRNAs were measured by real-time PCR. Error bars are s.d. of triplicates. WT: wild-type of Brca1 plasmid, Mu: Brca1-BRCT mutant (M1775R, point mutation in BRCT domains).