Phospho brca1

Whole-cell lysates were harvested using radioimmunoprecipitation assay buffer with a protease inhibitor cocktail and the PhosSTOP phosphatase inhibitor (Roche, Basel, Switzerland), and protein concentrations were determined using the BCA assay (Pierce, Dallas, TX, USA). Protein samples were then subjected to 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and blotted onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% dry skim milk and then incubated with the following primary antibodies at 4 °C overnight: anti-Flag (Sigma-Aldrich, St. Louis, MO, USA), His, phospho-ATR (Ser428), phospho-ATM (Ser1981), phospho-Chk1 (Ser345), phospho-Chk2 (Thr68), phospho-BRCA1 (Ser1524), phospho-p53 (Ser15), PARP, cleaved PARP, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-9, cleaved caspase-9 (Cell Signaling Technology, Danvers, MA, USA). After washing, the blots were incubated with horseradish peroxidase-labeled secondary antibodies (Cell Signaling Technology) and detected by enhanced chemiluminescence (EMD Millipore). GAPDH (Cell Signaling Technology) served as an endogenous loading control. Full gel images for all western blot experiments are provided in Supplementary Fig. 6.

Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F-12) and fetal bovine serum were purchased from Gibco-Invitrogen (Grand Island, NY, USA). Propofol and Dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Akt inhibitor (wortmannin) and AMPK inhibitor (dorsomorphin) were purchased from Selleck. Akt activator (IGF-1) and AMPK activator (AICAR) were purchased from RD Systems. Antibodies against FoxO1 and Nrf2 were purchased from Abcam. Antibodies against BAX, phospho-ATM (Ser1981), phospho-ATR (Ser428), γ-H2AX (Ser139), phospho-CHK1 (Ser345), phospho-CHK2 (Thr68), phospho-P53 (Ser15), phospho-BRCA1 (Ser1524), Akt, phospho-Akt (Thr308), IRS-1, phospho-IRS-1 (Ser636/639), AMPK, phospho-AMPK (Thr172), and phospho-FoxO1 (Ser256) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against NOX2, SIRT1, Cat, SOD1, Histone-H3, and GAPDH were purchased from Proteintech (Chicago, IL, USA).