Anti brca1

The lentivirus-mediated shNFBD1 and shControl were purchased from Genechem, Shanghai, China. Hoechst 33342 were purchased from Beyotime Institute of Biotechnology (Nantong, China).The antibodies used in this study were anti-NFBD1 and RPA1 (Abcam, UK); anti-RAD51, anti-BRCA1 and anti-BRCA2 (Santa Cruz Biotechnology, USA); anti-γ-H2AX and anti-phospho-histone H3 (Ser10) (Cell Signaling Technology, Danvers, MA, USA).

Cells were cultured on coverslips and were treated with 10 μM VP-16 for the indicated times. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, and were subsequently permeabilized in 0.5% Triton X-100 solution for 10 min. After being blocked by 5% BSA, cells were incubated with primary antibodies overnight and subsequently secondary antibodies for 1 h. Coverslips were then mounted using DAPI containing anti-fade. Images were captured using a Leica SP8 Laser confocal optical imaging platform. Images were processed using Leica Application Suite X. The percentage of cells carrying indicated foci was calculated after analyzing three independent experiments. Approximately 150 cells were counted for each sample. Antibodies used for immunofluorescent staining are as follows: anti-gamma-H2AX (Abcam; Mouse; ab22551; 1:100 dilution), anti-Rad51(Abcam; Rabbit; ab133534; 1:400 dilution), anti-Brca1(Santa Cruz Biotechnology; Mouse; sc-6954; 1:100 dilution), and anti-FLAG (Cell Signaling Technology; Rabbit; #14793; 1:200 dilution). The following secondary antibodies were used: Alexa Fluor 488 Goat Anti-Mouse (Life Technologies; A-11008; 1:1000) and Alexa Fluor 594 Goat Anti-Rabbit (Life Technologies; A-11012; 1:1000).

Protein from cells or tissues was extracted standardization with a BCA kit (Pierce, Rockford, IL, USA). Then, protein samples (40 μg) were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were then incubated with the following primary antibodies after blocking in 5% nonfat milk: anti-BRCA1 (1:600), anti-p-Smad3 (1:800), anti-Smad3 (1:800), anti-TGF-β1 (1:1000) and anti-β-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were then incubated with secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature before the chemiluminescence was measured. The Quantity One program (Bio-Rad, Hercules, CA, USA) was used to measure the intensity of the protein bands.

SIRT1 selective inhibitor (Ex527) was purchased from Sigma (Cat No. E7034) and diluted in dimethyl sulfoxide (DMSO) to 20 mg/mL as stock solution. Commercial antibodies were used to detect anti-acetylated lysine (Rockland, rabbit polyclonal, Cat No. 600-401-939), anti-GAL4 DNA binding domain (Millipore, rabbit polyclonal, Cat No. 06–262), anti-BRCA1 (Santa Cruz, mouse monoclonal, Cat No. sc-6954), anti-FLAG (Sigma, mouse monoclonal, Cat No. F3165), anti-HA (Sigma, rabbit polyclonal, Cat No. H6908/ mouse monoclonal, Cat No. H3663), and anti-β-actin (Sigma, mouse monoclonal, Cat No. A2228). Homemade antibodies against SIRT1, KAP1, phospho-KAP1 (Ser824), and 53BP1 were raised by immunizing rabbits with SIRT1-peptide (724–747 a.a), GST-KAP1 (697–835 a.a), phospho-KAP1 (824)-peptide (GAGLS(pS)QELSG), and GST-53BP1 (338–671 a.a).

As primary antibodies, anti-DDX11 (Santa Cruz #sc-271711) (1:1,000), anti-BRCA1 (Santa Cruz #sc-6954) (1:1,000), anti-BRCA2 (ab123491) (1: 1000), anti-Chk2 (Santa Cruz #sc-17747) (1:1,000), anti-pChk2 (pT68) (cell signaling technology #2661) (1:1,000), anti-DNA PKcs (Epitomics #1579–1) (1:1,000), anti-DNA PKcs (pS2056) (Epitomics #3892–1) (1:1,000), anti–γ-H2AX (Millipore #05–636) (1:500), anti-53BP1 (Novus Biologicals #NB100-304) (1:1,000), anti-MAD2B (REV7) (BD Biosciences 612266) (1:1,000), anti-RAD51 (Santa Cruz #sc-17747) (1:50), anti-RPA2 (RPA32) (Thermo Fisher #MA1-26418), and α-tubulin (Santa Cruz #8035) (1:5,000) were used. As secondary antibodies, anti-mouse HRP-linked (1:5,000 cell signaling technology), anti-rabbit HRP-linked (1:5,000 cell signaling technology), and Alexa Fluor 488 anti-mouse (immunofluorescence 1:400) Invitrogen, Alexa Fluor Cy3-conjugated anti-rabbit (immunofluorescence 1:400) Invitrogen were used.