Horseradish peroxidase hrp linked secondary antibody

Cells were lysed in boiling buffer (10% SDS, 0.5 M Tris–HCl pH 6.8) and centrifuged at 20,000×g for 30 minutes; 20 μg of protein were separated on 7.5-15% SDS-PAGE and transferred onto 0.45-μm nitrocellulose membranes (GE Healthcare Europe, Milan, Italy). Blots were stained using standard procedures and signals were revealed by a chemiluminescence reagent (Euroclone, Milan, Italy). Horseradish peroxidase (HRP)-linked secondary antibodies, anti-phospho-Ataxia Telengectasia (Ser 1981) (ATM), anti-ATM, anti-phospho-p53, anti-p53 and anti-vinculin were from Cell Signaling Technology (Beverly, USA); anti-phospho-Histone H2A.x (Ser 139) was from Millipore (Temecula, CA) and anti-Histone H2A.x was from GeneTex (San Antonio, Texas). Three independent experiments were performed.

Cells were washed with PBS and lysed with lysis buffer (62.5 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol). Cell lysates were centrifuged at 20, 000 g for 10 min at 4°C, and proteins in the supernatants were quantified. Protein extracts were equally loaded onto an 8% to 14% SDS–polyacrylamide gel, electrophoresed, and transferred to nitrocellulose membrane (Amersham Bioscience, Buckinghamshire, UK). The blots were stained with 0.2% Ponceau S red to ensure equal protein loading. After blocking with 5% nonfat milk in PBS, the membranes were probed with antibodies against poly (ADP [adenosine diphosphate]–ribose) polymerase (PARP; 1∶500; Santa Cruz Biotech, Santa Cruz, CA), PRDX 1, PRDX 2, PRDX 3, PRDX 4, PRDX 5, PRDX 6, and Sirt1 (1∶1000; Santa Cruz Biotech), and followed by horseradish peroxidase (HRP)–linked secondary antibodies (Cell Signaling). The signals were detected by Chemiluminescence phototype-HRP kit (Cell Signaling) according to manufacturer’s instructions. As necessary, blots were stripped and re-probed with anti-actin or anti-tubulin antibodies (Oncogene, Fremont, CA) as an internal control. The signal intensity of the bands was quantified using Quantity One Version 4.4.0 (Bio-Rad). All experiments were repeated three times.

Cells or tissues were digested, washed with cold PBS, and lysed in 1x RIPA buffer containing phenylmethanesulfonyl fluoride (PMSF) on ice to extract protein. After sonication, the proteins were separated by SDS-PAGE and electrically transferred to a polyvinylidene difluoride membrane with a pore size of 0.45 μm (Millipore, Bedford, MA, United States). Each membrane was blocked with 5% BSA solution and incubated with appropriate primary antibodies diluted with TBS/T containing 1% BSA overnight at 4°C, followed by reaction with horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling Technology, Beverly, MA, United States) at room temperature for 1 h before chemiluminescence detection. The antibodies used in western blotting are listed in Supplementary Table S3.

Cells were homogenized with RIPA lysis buffer containing a protease inhibitor mixture (Thermo Fisher Scientific). The homogenates were centrifuged at 15,000 × g at 4 °C for 10 min, and the supernatants were collected. Approximately 20 μg of total protein extract was subjected to 10% SDS-PAGE gels and then transferred onto polyvinylidene fluoride membranes (Millipore, Temecula, CA, USA). The membranes were blocked with 1% BSA in TBS for 1 h and then probed with anti-SOX10 antibody (Abcam) or other primary antibodies (Abcam) at 4 °C overnight, followed by reaction with horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) at room temperature for 1 h. The membranes were then imaged on the ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Information of the antibodies is listed in Supplementary Table 3.

Whole-cell extracts were prepared by employing cell lysis buffer (9803) from Cell Signaling Technology with protease and phosphatase inhibitor cocktails I and II (Cat. # BP-479 and BP-480, Boston BioProducts, MA, USA). Protein samples (30–60 μg) were loaded and separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membrane was blocked by 5% nonfat powdered milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 30 min and then probed with different antibodies in 4% powdered milk in TBST, as previously described [59 (link)]. The membrane was washed extensively with TBST, and then incubated with the appropriate horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling Technology, Beverly, MA, USA). Signals were visualized with an enhanced chemiluminescence detection system (GE Healthcare, Pittsburgh, PA, USA) and quantified by densitometry. Equal loading of protein was verified by immunoblotting with the anti-β-actin antibody.

Protein samples (20 μg) from total liver lysates were resolved via 10% SDS-PAGE gels (Bio-Rad, San Diego, CA, USA) and transferred to nitrocellulose membranes (GE Healthcare, Chicago, IL) using the Mini Trans-Blot Cell Unit (BioRad) by wet electroblotting (100 V, 45 min). The membranes were then blocked with 5% nonfat milk (in Tris-buffered saline [TBS] + 0.01% Tween) for 1 h and incubated with primary antibodies: anti-β-hydroxybutyryllysine (1:1000; PTM-1201; PTM Biolabs, China) or anti-β-actin (1:1000; #4970; Cell Signaling) overnight at 4 °C. Blots were visualized using horseradish peroxidase (HRP)-linked secondary antibodies (1:3,000; Cell Signaling Technology) and an ECL kit (Millipore Corporation, Billerica, MA). Membranes were scanned on a Sapphire Biomolecular Imager (Azure Biosystem, Dublin, CA). Protein density values were assessed and calculated using ImageJ software (version 1.47; National Institutes of Health, Bethesda, MD). The expression for the protein of interest was standardized to β-actin levels.