The experimental methods were previously described57 (link). The primers for mouse Yap1 (ID 15928514a1) were designed based on the online PrimerBank database (Harvard Medical School, Boston, MA; http://pga.mgh.harvard.edu/primerbank ). The mouse anti-YAP1 antibody (1:100, cat # sc-101199) was purchased from Santa Cruz Biotechnology, and mouse anti-β-actin antibody (1:1000, cat #A2228) was purchased from Sigma.
Mouse anti β actin antibody
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Macao, China, France
The Mouse anti-β-actin antibody is a laboratory reagent used for the detection and analysis of the β-actin protein. β-actin is a highly conserved cytoskeletal protein found in all eukaryotic cells and is commonly used as a loading control or reference protein in various experimental techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (10 mentions)
Protease inhibitor cocktail, by Merck Group (7 mentions)
Odyssey infrared imaging system, by LI COR (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (4 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Tween 20, by Merck Group (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Cytiva (3 mentions)
Glycerol, by Merck Group (3 mentions)
Glycine, by Merck Group (3 mentions)
Trizma base, by Merck Group (3 mentions)
Goat anti mouse and goat anti rabbit horseradish peroxidase conjugated igg, by Jackson ImmunoResearch (3 mentions)
Las 4000, by Cytiva (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Amersham ecl prime, by Cytiva (3 mentions)
217 protocols using mouse anti β actin antibody
1
Mouse Yap1 Gene Expression Analysis
2
Signaling Pathway Exploration in Gastric Cancer
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Vero cells and human gastric cancer cells (MKN1, MKN28, and MKN74) were incubated in 10-cm dishes at 2 × 106 cells per dish at 37°C. After 24 hr of incubation, cells were infected with PBS (–) and T-01 (Vero cells: at MOI of 0.01, human gastric cancer cells: at MOI of 0.1) and T-SOCS3 (Vero cells: at MOI of 0.01, human gastric cancer cells: at MOI of 0.1). After 20 hr of incubation at 39.5°C, proteins (30 μg) were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membrane (Bio-Rad) and blotted for 2 hr with additive monoclonal mouse anti-SOCS3 antibody (diluted 1:1000, R&D systems), anti-pSTAT3 antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA) or with mouse anti-β-actin antibody (diluted 1:2000, Sigma) for an hour. After the membrane rinsed out, a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (diluted 1:4000, GE healthcare, Piscataway, NJ, USA) was blotted and rinsed. And the, the membrane was exposed to enhanced luminol-based chemiluminescent (ECL) Plus (GE healthcare, Japan).
3
Quantitative Western Blot Analysis
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Cells or skin samples crushed in liquid nitrogen were solubilized at 4°C in lysis buffer (0.5% sodium deoxycholate, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 100 μg/ml phenylmethylsulphonyl fluoride, and protease inhibitor cocktail). Five micrograms of protein were separated on SDS-polyacrylamide gels and transferred onto polyvinylidine fluoride membranes (Bio-Rad, Hercules, CA USA). Non-specific antibody binding was blocked by incubating the membranes in 5% w/v non-fat milk powder in TBS-T (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% v/v Tween-20). The membranes were incubated with rabbit anti-POSTN antibody (Abcam, Cambridge, UK) diluted 1:1000 overnight at 4°C or with mouse anti-β-actin antibody (Sigma- Aldrich, St Louis, MO, USA) diluted 1:10,000 overnight at 4°C. Then, the membranes were washed 3 times in TBS-T for 5 min. Finally, the membranes were incubated with either HRP-conjugated anti-mouse or anti-rabbit at a dilution of 1:10,000 for 60 min at room temperature. Protein bands were detected using the ECL Plus kit (Thermo Scientific, Rockford, IL USA). The intensity of the bands was quantified using NIH image J software.
4
Oxidative Stress Signaling Pathways
5
Intestinal Crypt Protein Analysis
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The intestinal crypts were homogenized and total protein was extracted using radioimmunoprecipitation assay (RIPA) buffer. Protein concentrations were determined using the bicinchoninic acid assay (BCA) method (ThermoFisher Scientific), normalized for all samples and separated on NuPAGE Novex 4–12% Bis-Tris gels (ThermoFisher Scientific). Subsequently, the proteins were transferred to polyvinylidene difluoride membranes (ThermoFisher Scientific), blocked with 5% skimmed milk in tris-buffered saline Tween (TBST), and incubated overnight with goat anti-C3/C3 fragments antibody (EMD Millipore) at a dilution 1:1000, rabbit anti-Ki67 antibody (Abcam) at a dilution 1:500, rabbit anti-Lgr5 antibody (ThermoFisher Scientific) at a dilution 1:1000, goat anti-C3aR1 antibody (Santa Cruz Biotechnology) at dilution a 1:500, and mouse anti-β-actin antibody (Sigma-Aldrich) at dilution a 1:5000 in TBST containing 5% skimmed milk. After three washes with TBST, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). Protein bands were visualized using a chemiluminescent ECL™ detection kit (GE Healthcare).
6
Western Blot Analysis of Cellular Stress Markers
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Cells were seeded and differentiated in Costar 25 cm2 flasks (Corning) at 1.2 million cells/well. After incubation, cells were scrapped in 150 μL lysis buffer (40 mM Tris pH 7.5, 50 mM potassium chloride, 1 mM EDTA, 1% sodium dodecyl sulfate) containing a protease inhibitor mixture (1 tablet in 2 mL water added at a 1/25 dilution; Hoffman-La Roche Ltd, Basel, Switzerland) and phosphatase inhibitors (sodium metavanadate 25 mM, 4-nitrophenyl phosphate 250 mM, β-glycerophosphate 250 mM, sodium fluoride 125 mM at a 1/25 dilution). Western blot analysis was performed as described in Notte et al24 (link) using a rabbit monoclonal anti-phosphorylated eIF2α antibody (119A11; Cell Signaling Technology, Inc., Danvers, MA, USA), a mouse anti-β-actin antibody (A5441; Sigma), a rabbit polyclonal anti-cleaved caspase 3 antibody (9662S; Cell Signaling), a rabbit polyclonal anti-caspase 7 antibody (9492; Cell Signaling), a rabbit polyclonal anti-caspase 4 antibody (4450S; Cell Signaling), a rabbit polyclonal anti-caspase 9 antibody (9502; Cell Signaling), a mouse monoclonal anti-caspase 8 antibody (9746; Cell Signaling), a mouse monoclonal anti-LC3 antibody (Nanotools GmbH, Munchen, Germany), or a mouse anti-p62 antibody (Abnova, Taipei, Taiwan).
7
ER Stress Pathway Protein Detection
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Anti-phospho-PERK antibody was obtained from Pierce (Rockford, IL, USA). The antibody for phospho-eIF2α was obtained from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-LC3B antibody, mouse anti-β-actin antibody, rabbit anti-GRP78 antibody, rabbit anti-ATF6 antibody, rabbit anti-ATF4 antibody, and rabbit anti-phospho-IRE1 antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Small interfering RNAs (siRNAs) targeting PERK and IRE1 were synthesized by Shanghai GenePharma Co. Ltd. WonderOrange ProteinQuantitationKit(US Everbright Inc, Suzhou,China).
8
Antibody Detection for Protein Analysis
9
Western Blot Analysis of WNV NS1 Protein
10
Western Blot Analysis of LC3B Protein Expression
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Cells cultured at 1 g and in RPM condition for 24 and 48 hours were lysed in RIPA buffer (Sigma-Aldrich). Samples were then clarified by centrifugation at 10000 rpm for 10 min. Equivalent amount of protein (10 μg) from each sample was electrophoretically resolved on 12.5% precast SDS-polyacrylamide gels (ExcelGel, GE Healthcare Biosciences) using horizontal apparatus (Pharmacia Biotech, Uppsala, Sweden). Then, separated proteins were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell) by a semidry system (Novablot, Pharmacia Biotech). Membranes were blocked with 3% nonfat milk in PBS and then were incubated (ON at 4°C) with the LC3B monoclonal antibody (1 : 2000; Sigma). After extensive washing with PBS containing 0.1% tween-20 (TBST), blots were incubated with 1 : 2000 dilution of HRP-conjugated secondary antibody (Amersham Biosciences) for 1 hour at RT. Immunopositive bands were detected with a chemiluminescence's detection system (GE Healthcare Biosciences). To check for equal loading of the gel, membranes were stripped and reprobed with mouse anti-β-actin antibody (1 : 20000, Sigma) and with anti-GAPDH antibody (1 : 1000, Cell Signalling Technology). Densitometric analysis was performed with the Quantity One software (BioRad Laboratories).
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