Anti gapdh 60004 1 lg

High performance cell lysate (P0013B, Beyotime, China) was added to the cells, then lysed for 20 min until the cells were completely ruptured. The supernatant protein was carefully aspirated after centrifugation at 13,300 rpm for 15 min. The proteins were separated by electrophoresis on 10% SDS-PAGE gel and transferred to PVDF membranes (Invitrogen, USA). After blocking with 5% bovine serum albumin (BSA) for 1 h, the cells were incubated with primary antibody overnight at 4 °C. Then it was incubated with HRP-labeled secondary antibody (Jackson) for 1 h, and finally the bands were detected by chemiluminescence. Information for primary antibodies: anti-wnt11 (ab31962, Abcam), anti-β-catenin (ab32572, Abcam), anti-GAPDH (60004-1-lg, Proteintech).

Immunoblotting was performed as previously described 16 (link). Anti-Aurora-A (D3E4Qz, #14475), anti-Acc 1 (#4190), anti-AKT (C67E7, #4691), anti-p-AKT (Ser473, #9271), and anti-PPARγ (C26H12, #2435) antibodies were purchased from Cell Signaling Technology (MA, USA). An anti-SLC7A5/LAT1 (#DF8065) antibody was purchased from Affinity. Anti-β-actin (66009-1-Ig), anti-GLUT4 (66846-1-lg), and anti-GAPDH (60004-1-lg) antibodies were purchased from Proteintech.

Total protein was extracted from cell lines using RIPA Lysis Buffer (Biosharp, China) containing a protease inhibitor and quantified using a BCA Protein Assay Kit (Biosharp, China) according to the manufacturer’s instructions. Western blotting was performed using a standard protocol. The primary antibodies were: anti-INPP4B (BM4609, Boster, USA), anti-PD-L1 (66248-1-lg, Proteintech), anti-p85 (660225-1-lg, Proteintech), anti-Phospho-AKT (66444–1-lg, Proteintech), anti-Bcl-2 (12789–1-AP, Proteintech), and anti-Cyclin D1 (60186–1-lg, Proteintech). In addition, an anti-GAPDH (60004–1-lg, Proteintech) was used as loading control.

Protein lysis from cells, exosomes and tissues were prepared and the protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA.). Protein Samples were separated by 10% or 12% SDS-PAGE gels and transferred to nitrocellulose filter membranes. The nitrocellulose filter membranes were blocked with 3% skim milk in tris buffered saline (TBS) containing 0.1% Tween-20 (TBST) at 4 °C overnight, and then incubated with primary antibodies followed by a horseradish peroxidase-conjugated secondary antibodies (washed with TBST three times before each operation, 5 min each time). Primary antibodies used were anti-LDLR (10785-1-AP, Proteintech), anti-GM130 (sc71166, Santa Cruz), anti-TSG101 (ab83, Abcam), anti-CD63 (ab134045, Abcam), anti-GAPDH (60004-1-lg, Proteintech), secondary antibodies used were anti-Rabbit (7074, CST) and anti-Mouse (7076, CST).

GFP-CtIP, full-length and truncated FLAG–CtIP were generously provided by Dr. Junjie Chen (MD Anderson Cancer Center, TX). FLAG–ASF1A was generously provided by Dr. Zhiguo Zhang (Columbia University, NY). Full-length and truncated FLAG–USP52 and Myc-USP52 were generously provided by Dr. Lei Shi (Tianjin Medical University, Tianjin, China). FLAG–USP52 S1003A and S469A, FLAG–CtIP K62R, K760R, K782R, and 2KR mutants were generated by site-directed mutagenesis (Stratagene). Flag–CtIP–T847E was purchased from Addgene. CHX, IgG agarose, anti-FLAG agarose, 3×FLAG peptide and ATM inhibitor KU55933 were purchased from Sigma Aldrich. Olaparib was purchased from Toronto Research Chemicals. Anti-USP52 (ab241505, 1:1000) was purchased from abcam; anti-CtIP (Thr847) (p1012-847, 1:2000), and anti-CtIP (Thr327) (p1012-327, 1:2000) were purchased from PhosphoSolutions; anti-RPA32 (sc-56770, 1:2000), anti-Ub (sc-8017, 1:2000) and anti-CtIP (sc-271339, 1:1000 for WB) were purchased from Santa Cruz; anti-FLAG (F1804, 1:2000) was purchased from Sigma; anti-pS345 Chk1 (2348, 1:1000), anti-Myc Tag (2276, 1:2000) and anti-SQ/TQ motif (9607, 1:1000) were purchased from CST; anti-CtIP (61141, 1:1000 for IF) was purchased from Active Motif; anti-GAPDH (60004-1-lg, 1:2000) was purchased from Proteintech; anti-Rad51 (GTX100469, 1:1000) was purchased from Genetex.

Resveratrol was purchased from Nanjing Spring & Autumn Biological Engineering Co., Ltd. (Nanjing, China). Anti-pAMPKα (Thr172) (40H9) and anti-Cav-1 (D46G3) were from Cell Signaling Technology. Anti-LC3 (NB600-1384) was from NOVUS. Anti-PPARγ (H-100) was from Santa Cruz Biotechnology. Anti-PGC1α (ab54481) was from ABcam. Anti-AMPKα (10929-2-AP), anti-Sirt1 (13161-1-AP) and anti-GAPDH (60004-1-lg) were from Proteintech (Wuhan, China).

Polyclonal anti-BCAT1 antibody (13640–1-ap; Protein Tech) was used for immunofluorescence staining. Western blotting was performed using the following antibodies: rabbit polyclonal anti-BCAT1 (13640–1-ap), anti-pS6K1 (ab59208; Abcam), rabbit monoclonal anti-p-mTOR (ab109268; Abcam), anti-mTOR (ab32028; Abcam), anti-S6K1 (ab32359; Abcam), and mouse monoclonal anti-GAPDH (60004–1-lg; Protein Tech).