Cells or tumor tissue samples were lysed with RIPA buffer (Beyotime) containing protease inhibitors cocktail (Selleck). After centrifugation, samples were loaded on to and separated using SDS/PAGE, then transferred on to PVDF membranes (Millipore). The membranes were blocked for 1 h at room temperature and incubated with the anti-β-actin (#3700, Cell Signaling Technology), anti-PARP1 (13371-1-AP, Proteintech) and anti-Caspase 3 (#9662, Cell Signaling Technology) antibodies overnight at 4°C. After washing, the blots were incubated with goat anti-rabbit (111-035-003, Jackson) or anti-mouse (115-035-003, Jackson) HRP-conjugated secondary antibodies and visualized using the Immobilon™ HRP Substrate Peroxide Solution (Millipore).
Anti mouse 115 035 003
Manufactured by Jackson ImmunoResearch
Sourced in United States
Anti-mouse (115-035-003) is a secondary antibody produced in donkey and affinity purified against mouse IgG. It is conjugated with horseradish peroxidase (HRP) and can be used for detection in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Goat anti rabbit 111 035 003, by Jackson ImmunoResearch (2 mentions)
Protease inhibitors cocktail, by Selleck Chemicals (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti parp1 13371 1 ap, by Proteintech (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Anti rabbit 111 035 003, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
F actin antibody, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Polyclonal rabbit anti ps214 tau, by Thermo Fisher Scientific (1 mentions)
Anti lc3, by Novus Biologicals (1 mentions)
Anti gfp, by Abcam (1 mentions)
Anti p62, by Abnova (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti sirt1, by Cell Signaling Technology (1 mentions)
10 protocols using anti mouse 115 035 003
1
Western Blot Analysis of Apoptosis Markers
2
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).
3
Immunostaining of cell signaling proteins
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SPANXB1 (#H00728695) and SH3GL2 (#H00006456) antibodies were purchased from Abnova. The CDH1 (#3195P), RAC-1 (#2465), FAK (#3285), Alpha-ACTININ (#6487), Snail (#3879), Slug (#9585), ZO-1 (#8193), Vinculin (#13901) and GFP (#2956) antibodies were purchased from Cell Signaling. F-actin antibody (#A12380) was obtained from Invitrogen. Anti-mouse ( #115-035-003) and rabbit (#111-035-003) secondary antibodies were obtained from Jackson Immunoresearch. DAPI (#62248) and Phalloidin (#A12379) were purchased from ThemoFisher.
4
Quantifying Protein Expression via Western Blot
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The amount of each protein under investigation was expressed as a ratio of the intensity of the protein band divided by the intensity of the GAPDH band (Santa Cruz Biotechnology anti-mouse SC-32233 1/4,000 and Jackson ImmunoResearch anti-mouse 115-035-003, 1/5,000) to account for variations due to loading, even though the amount of protein loaded was previously quantified using the standard Bradford assay. This ratio was normalized by dividing it by the ratio obtained, in the same Western blot, from a standard control mouse. The same standard mouse was used to normalize all Western blots across different backgrounds and age groups. Each sample was run in triplicate and the average normalized ratio was used for comparison. Image J was used to carry out densitometry calculations.
5
Antibody and Reagent Preparation for In Vitro Experiments
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The primary antibodies used in this study are listed in Supplementary Table 1 . The secondary antibodies were obtained from Jackson Immuno Research Laboratories Inc. (West Grove, PA, United States) (anti-mouse #115-035-003; anti-rabbit# 211-002-171). The C-peptide was synthesized by Peptrone (Daejon, South Korea). A palmitic acid (PA) solution was prepared using 20 mM PA in 150 mM NaCl with 5% bovine serum albumin. Okadaic acid (OA) was dissolved in DMSO. A list of the reagents used in this study is provided in Supplementary Table 2 .
6
Tau and Autophagy Protein Detection
7
Antibody Panel for Cell Signaling
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The anti-SIRT1 (# 2496), anti-Atg5 (# 2630), anti-ULK1 (# 6439), anti-LC3 (# 4108), anti-PARP (# 9542), anti-Bak (# 12105), anti-Bax (# 2772), anti-Puma (# 4976), anti-Noxa (# 14766), anti-Bcl2 (# 15071), anti-c-Flip (# 56343), anti-c-Myc (# 5605), HA-tag (# 3724), and anti-cleaved caspase-3 (# 9661) antibodies were purchased from Cell Signaling Technology, Inc (Beverly, MA, USA). The anti-acetyl-c-Myc (# ABE26) antibody was from Millipore Corp. (Temecula, CA, USA). The anti-Atg7 (# NB110-55474) antibody was obtained from Novus Biologicals (Centennial, CO, USA). The anti-NOX4 (# SC-30141) antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The antisera to tNOX used for immunoblotting were generated as described previously (Chen et al., 2006 (link)). The commercially available anti-ENOX2 (# 10423–1-AP) antibody and anti-β-actin (# 60008–1-Ig) antibodies were from Proteintech (Rosemont, IL, USA) was used for immunoprecipitation. The anti-mouse (# 115-035-003) and anti-rabbit IgG (# 111-035-003) antibodies were purchased from the Jackson ImmunoResearch Laboratories Inc, (West Grove, PA, USA).
8
Protein Extraction and Western Blot Analysis
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Proteins were extracted using RIPA lysis buffer containing a protease inhibitor cocktail (Sigma, St. Louis, MO, U.S.A.) and a phosphatase inhibitor cocktail (Roche, Mannheim, Germany). After quantitation using the Bradford method, protein lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore). The membranes were blocked with nonfat milk for 1 h and incubated with primary antibodies, which are listed in Supplementary Table S1, overnight at 4°C. They were then incubated with goat anti-rabbit (111-035-003, Jackson) or anti-mouse (115-035-003, Jackson) HRP–conjugated secondary antibodies. Protein bands were visualized by enhanced chemiluminescence (Millipore). β-actin was used as a loading control.
9
Protein Extraction and Immunoblotting Protocol
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Proteins were extracted using a lysis buffer containing 12mM Hepes, 300mM mannitol, 1mM EGTA, 1mM EDTA, 1%Triton X-100, and 0.1% SDS adjusted to pH 7.6, in the presence of 1mM n-ethylmaleimide, 1mM phenylmethanesulfonyl fluoride and protease inhibitor cocktail (P8340, Sigma-Aldrich). Protein extracts were clarified by centrifugation and then quantified using the Pierce BCA Protein Assay kit (ThermoFisher). 20µg of protein extracts were subjected to SDS-PAGE and then transferred onto a PVDF membrane. Immunoblotting was carried out using either anti-CaSR antibody (ImmunoGenes) or anti-Tubulin antibody (T5168, Sigma-Aldrich). HRP-conjugated antirabbit (111-035-003, Jackson ImmunoResearch, USA) and anti-mouse (115-035-003, Jackson ImmunoResearch) were used as secondary antibodies. Chemiluminescence was detected by a ChemiDoc MP Imaging System (Biorad, USA).
10
Western Blot Analysis of Key Cellular Proteins
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Cells or tissues were lysed with RIPA buffer (Beyotime) containing protease inhibitors cocktail (Selleck). Protein samples were loaded onto and separated using SDS-PAGE, then transferred onto polyvinylidene fluoride membranes (Milliore). The membranes were blocked for 1 h at room temperature and incubated with the anti-LC3B (#3868, Cell Signaling Technology), anti-ACTB (#3700, Cell Signaling Technology), anti-TCF4 (#2565, Cell Signaling Technology), anti-β-catenin (#8480, Cell Signaling Technology), anti-Bcl2 (12789-1-AP, Proteintech), or anti-Beclin1 (#3495, Cell Signaling Technology) antibodies overnight at 4 °C. After washing, the blots were incubated with goat anti-rabbit (111-035-003, Jackson) or anti-mouse (115-035-003, Jackson) horse radish peroxidase-conjugated secondary antibodies and visualized using the Immobilon™ horse radish peroxidase substrate peroxid solution (Millipore).
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