BMDMs and RAW264.7 cells were lysed using 1%-Triton X-100 in PBS (pH 7.4), EDTA-free protease inhibitor cocktail (Roche Diagnostics) and 10 mM of sodium pyrophosphate (Sigma) in PBS. Cell lysates were subjected to 10–15% SDS-PAGE. Separated proteins were transferred onto 0.2 μm nitrocellulose membrane (GE Health care) and blotted with specific antibodies. SMAD-2, phospho-SMAD2 and LC-3 antibodies were purchased from Cell signaling. Beclin-1 antibody was purchased from Abcam. RSV fusion (F) protein antibody was obtained from FDA (Dr. Judy Beeler). β-actin antibody was purchased from Bethyl Laboratories. For some experiments, protein bands from Western blot were quantified using Image Lab Software (Bio-Rad).
β actin antibody
Manufactured by Fortis Life Sciences
Sourced in United States
The β-actin antibody is a protein-specific antibody that recognizes the β-actin protein, which is a key component of the cytoskeleton in eukaryotic cells. It is commonly used in various laboratory applications to detect and quantify the expression levels of the β-actin protein.
Automatically generated - may contain errors
Lab products found in correlation
Immunochemiluminescent reagents, by GE Healthcare (3 mentions)
0.2 μm nitrocellulose membrane, by GE Healthcare (2 mentions)
Lamin b1 antibody, by Bioworld Technology (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Free protease inhibitor cocktail, by Roche (1 mentions)
Sodium pyrophosphate, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Beclin 1 antibody, by Abcam (1 mentions)
Parp antibody, by Santa Cruz Biotechnology (1 mentions)
Streptomycin, by Welgene (1 mentions)
12 o tetradecanoylphorbol 13 acetate tpa, by Merck Group (1 mentions)
Rpmi 1640 medium, by Welgene (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Ht29 human colorectal cancer cell line, by American Type Culture Collection (1 mentions)
Lps escherichia coli 0127 b8, by Merck Group (1 mentions)
Il 1β, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Chemidoc xrs software image lab 5, by Bio-Rad (1 mentions)
Anti rfp antibody, by Thermo Fisher Scientific (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
8 protocols using β actin antibody
1
Immunoblotting Analyses of BMAM and RAW264.7 Cells
2
Immunoblotting Analysis Protocol
3
Colorectal Cancer Cell Line HT-29 Cultivation
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The HT-29 human colorectal cancer cell line was purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Roswell Park Memorial Institute medium (RPMI) 1640 medium supplemented with 10% fetal bovine serum (WelGene, Daegu, Korea) and 100 µg/mL streptomycin (WelGene) at 37 °C in a 5% CO2 atmosphere. RPMI was purchased from WelGene, PGA was purchased from Bioleaders (Daejeon, Korea), and 12-O-tetradecanoylphorbol-13-acetate (TPA) was purchased from Sigma (St. Louis, MO, USA). TUNEL and Annexin V assay kits were purchased from Millipore (Darmstadt, Germany). Antibodies against phospho-AMPK (Thr172), phospho-ERK (Thr202/Tyr204), phospho-Akt (Thr308), caspase 3, and AMPK from Cell Signaling Technology (Beverly, MA, USA). COX-2 and iNOS antibodies were purchased from Cayman (Ann Arbor, MI, USA). PARP antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-Actin antibody was purchased from Bethyl Laboratories (Montgomery, TX, USA).
4
Signaling Pathways Modulation Assay
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Dexamethasone, IL-1β and LPS (Escherichia coli 0127:B8) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein assay kit was purchased from Bio-Rad Lab. (Hercules, CA, USA). Antibodies against mitogen-activated protein kinases (MAPKs), signal transducer and activator of transcription 3 (STAT3), phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727) and several transcription factors were purchased from Cell Signaling Technologies (Danvers, MA, USA). β-Actin antibody was purchased from Bethyl Laboratories, Inc (Montgomery, TX, USA). Lamin B1 antibody was purchased from Bioworld technology, Inc (Minneapolis, MN, USA).
5
Western Blot Analysis of Signaling Proteins
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A549 cells were lysed using 1%-Triton X-100 (pH 7.4), EDTA-free protease inhibitor cocktail (Roche Diagnostics) in PBS. Cell lysates were subjected to SDS-PAGE and separated proteins were transferred onto 0.2 μm nitrocellulose membrane (GE Health care) and blotted with specific antibodies. β-catenin and FLAG antibodies were purchased from Sigma-Aldrich. LRP5 antibody was obtained from Cell signaling. β-actin antibody was purchased from Bethyl Laboratories. HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A). An anti-RFP antibody was purchased from Invitrogen. Western blots were quantified using ChemiDoc™ XRS + software Image Lab 5.1 (BioRad).
6
Protein Extraction and Western Blotting
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Protein lysates were obtained by collecting cells in a lysis buffer (20 mM HEPES, 250 mM NaCl, 2 mM EDTA, 1% SDS, 10% glycerol, 50 mM NaF, 0.1 mM hemin chloride, 5 mM NEM, 1 mM PMSF and 10 mg/mL leupeptin and aproptinin) [6] (link), [47] (link), [48] (link) followed by sonication. Protein concentration was performed using the BCA assay (Thermo Scientific). Equal amounts of protein extract (9–15 µg depending on experiment) from each lysate were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane as described. The blots were blocked overnight with 5% nonfat dry milk and incubated with specific antibodies. The antibodies used are detailed in Table 2 . After incubation with the appropriate secondary antibody, signals were detected using immunochemiluminescent reagents (GE Healthcare, Piscataway, NJ). Equal protein loading in each lane was confirmed with a β-actin antibody (#A300-491, Bethyl).
7
Anti-inflammatory Compound K Effects
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2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride (AMT) was purchased from Tocris Cookson Ltd. (Avonmouth, Bristol, UK). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dexamethasone, interleukin (IL)-1β, and lipopolysaccharide (LPS, Escherichia coli 0127:B8) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Compound K-enriched fraction (5%) was obtained from Fleton Natural Products Co. (Chengdu, China). The protein assay kit was purchased from Bio-Rad Lab (Hercules, CA, USA). All antibodies relating to MAPK and nuclear transcription factor-κB (NF-κB) signaling were purchased from Cell Signaling Technologies (Dancers, MA, USA). β-actin antibody was obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA). Lamin B1 antibody was purchased from Bioworld technology (Minneapolis, MN, USA).
8
Immunoblotting Analysis Protocol
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Standard protocols were used to perform all immunoblotting analyses (described in [13 (link), 24 (link)]). After incubation with appropriate secondary antibody, signals were detected using immuno-chemiluminescent reagents (GE Healthcare, Piscataway, NJ). β-actin antibody (#A300–491, Bethyl) was used as a protein loading control. See Additional file 2 : Supplementary Methods for detailed protocol. All the antibodies used for immuno-blotting has been listed in the Additional file 2 : Table S1.
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