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5 protocols using anti rabbit 111 035 003

1

Protein Extraction and Immunodetection

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Total protein extracts were obtained after lysing cells grown in monolayer in lysis buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCI pH 8.0, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS and supplemented with protease inhibitors. Protein concentration was determined by the Bradford method using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Primary antibodies used included anti-HMGB1 antibody [EPR3507] (ab79823), anti-Ube2N/Ubc13 antibody [EPR5162] (ab109286), anti-MyD88 antibody (ab2064), anti-TRAF6 antibody [EP591Y] (ab33915), anti-TLR4 antibody [76B357.1] (ab22048), and anti-SARM antibody (ab115561). All antibodies were purchased from Abcam (Cambridge, United Kingdom). The following secondary antibodies were used: anti-mouse (115-035-003) or anti-rabbit (111-035-003) (Jackson ImmunoResearch, West Grove, PA, USA). Peroxidase activity from secondary antibodies was assessed using the ECL kit (Amersham Biosciences, GE Healthcare, Chicago, IL, USA). Signals were quantified using ImageJ software (National Institutes of Health, Maryland, USA).
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2

Comprehensive Antibody Validation Toolkit

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Antibodies against β-Actin (AC026, 1:10000), NUDT1 (A13330, 1:1000 for immunoblots, 1:200 for immunoprecipitation), NOX4 (A11274, 1:1000), H3 (A2348, 1:1000), NCL (A5904, 1:1000) were obtained from ABclonal. PLK1 (4513 S, 1:1000), Phospho-PLK1 (Thr210) (5472 T, 1:1000), MYC (13987, 1:1000 for immunoblots), cleaved-Caspase 3 (Asp175) (9661 S, 1:1000 for immunoblots, 1:100 for IHC) were obtained from Cell Signaling technology. Flag-tag (F1804, 1 μg for immunoprecipitation) was obtained from Sigma–Aldrich. N-MYC (sc-53993, 1:1000 for immunoblots, 4 μg for ChIP) and PCNA (sc-56, 1:2000 for IHC) were obtained from Santa Cruz Biotechnology. 8-oxo-dG (AB5830, 1:300 for IHC) and γH2AX (05-636, 1:200 for IHC, 1:100 for IF, 1:1000 for immunoblots) were obtained from Millipore. HRP-conjugated goat anti-mouse (115-035-003, 1:5000) and anti-rabbit (111-035-003, 1:5000) secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. The Phospho-NUDT1 S121 antibody was generated by Dia-An Technology by immunizing rabbits with phosphorylated S121 peptides (PDD-(phospho)S-YWF) conjugated with carrier protein keyhole limpet hemacyanin.
Other chemicals include 4-OH Tamoxifen, Tetracycline HCl, and TH287 (Selleck Chemicals); Acetylcysteine, BI6727, MG132, Pomalidomide and SBE-β-CD (MCE); CHX (Sigma–Aldrich); DAPI (Yeasen); Alexa488-conjugated avidin (Invitrogen).
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3

Western Blot Antibody Panel Analysis

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The detailed procedures for western blot were described previously21 (link). The primary antibodies used in this study were monoclonal antibodies against p21 (#2946, Cell Signal, Beverly, MA, USA,), p27 (#3698, Cell Signal), CDK4 (cell signal, #2906, Beverly, MA, USA), CDK6 (cell signal, #3136, Beverly, MA, USA), cyclin D3 (cell signal, #2936, Beverly, MA, USA), NGAL(#PAB9543, Abnova Corporation, Taipei, Taiwan). The secondary antibodies (1:5000) were anti-rabbit (111-035-003, Jackson Immunoresearch, West Grove, PA, USA) or anti-mouse secondary antibodies (Zymed 81–6520). The blots were detected using ECL reagents (WBKLS0500, Millipore, Billerica, MA, USA). Membranes were detected by VersaDoc™ Imaging System (Bio-Rad, Hercules, CA, USA) for analysis.
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4

Western Blot Analysis of Phospho-p65 and Total p65

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After treatments, cells were lysed by scraping in RIPA buffer (ThermoFisher, cat no. PI89901) containing 1 mM PMSF, 1 mM NaVO4, 1 mM dithiothreitol, and 1 × protease inhibitor cocktail. Equal amounts of lysates (20-30 μg of total protein) were run on 10% SDS-PAGE gels, after which protein was transferred to nitrocellulose membranes (BioRad, Hercules, CA). Membranes were blocked in 5% BSA/Tris-buffered saline-Tween 20 (TBS-T) for one hour at room temperature prior to probing with primary antibodies overnight at 4 °C. Primary antibodies included anti-phospho-p65 (Ser 536, clone 93H1, #3033) and anti-p65 (clone D14E12, #8242) from Cell Signaling Technology (Danvers, MA), and anti-vinculin (clone hVIN-1, #V9131) from Sigma. After probing with primary antibodies, membranes were washed three times in TBS-T and then probed with the appropriate horseradish peroxidase-conjugated secondary antibodies (anti-mouse (#115-035-003) or anti-rabbit (#111-035-003) from Jackson ImmunoResearch). Then, the membranes were washed four times in TBS-T and developed using Clarity Western ECL substrate (BioRad, #1705060). Membranes were visualized using a BioRad ChemiDoc MP system (BioRad, Hercules, CA).
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5

Western Blot Antibody Detection Protocol

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The detailed procedures for Western blot were described previously [31 (link)]. The primary antibodies used in this study was monoclonal antibodies against LCN2 (#PAB9543, Abnova Corporation, Taipei, Taiwan). The secondary antibodies (1:5000) were anti-rabbit (111-035-003, Jackson Immunoresearch, West Grove, PA, USA) or anti-mouse secondary antibodies (Zymed 81-6520, San Francisco, CA, USA). The blots were detected using ECL reagents (WBKLS0500, Millipore, Billerica, MA, USA). Membranes were detected by VersaDocTM Imaging System (Bio-Rad, Hercules, CA, USA) for analysis.
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