Human gingival fibroblasts were starved in serum-free medium overnight before being stimulated for 30 min with salivary pellet that had been washed four times. Whole human saliva, tumor necrosis factor alpha (TNF-α, 10 ng/mL) and Interleukin (IL)-1 (10 ng/mL) served as positive controls, and serum-free medium as the negative control. Cell extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, GE Healthcare, General Electric Company, Fairfield, CT). Primary antibody binding was accomplished with phospho-NF-kB p65 and β-actin antibodies (Cell Signaling Technology, Danvers, MA). Secondary antibody bindings were detected by near-infrared absorbing dyes with the appropriate imaging system (LI-COR Biosciences, Lincoln, NE).
β actin antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Japan, Germany, Canada
The β-actin antibody is a primary antibody that specifically binds to the β-actin protein, a ubiquitously expressed cytoskeletal protein. It is commonly used as a loading control in western blotting experiments to normalize protein expression levels across samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (17 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Protease inhibitor cocktail, by Merck Group (8 mentions)
Protease inhibitor cocktail, by Roche (7 mentions)
Ripa buffer, by Thermo Fisher Scientific (7 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (7 mentions)
Bcl 2, by Cell Signaling Technology (6 mentions)
Ripa buffer, by Cell Signaling Technology (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Clarity western ecl substrate, by Bio-Rad (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Chemidoc mp imaging system, by Bio-Rad (5 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (4 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
N cadherin, by Cell Signaling Technology (4 mentions)
Erk1 2, by Cell Signaling Technology (4 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (4 mentions)
302 protocols using β actin antibody
1
Gingival Fibroblasts Activation by Saliva
2
S1PR1 Agonist CYM5442 Signaling
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The S1PR1 specific agonist CYM5442 was purchased from Sigma-Aldrich (MO, USA). Antibodies against ICAM1, total p65 subunit of NF-κB were purchased from Santa Cruz Biotechnology (TX, USA). Anti-p-p65 subunit, β-arrestin 2, and β-actin antibodies were obtained from Cell Signaling (MA, USA).
3
Platelet Aggregation Signaling Assay
4
Ginseng Fractions Modulate Macrophage Inflammation
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KRG, the NSF of KRG, and the SF of KRG were provided by the Korea Ginseng Cooperation (Daejeon, Korea). Detailed phytochemical profiles of these fractions are given in the Supplementary Information as previously reported [32 (link)]. Murine bone marrow–derived macrophage RAW264.7 cells were obtained from the American Type Culture Collection (Rockville, MD). Fetal bovine serum, Roswell Park Memorial Institute (RPMI) 1640, and antibiotics (penicillin and streptomycin) were purchased from Gibco (Grand Island, NY), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma Chemical Co. (St. Louis, MO). TRIzol reagent was purchased from Molecular Research Center (Montgomery, OH). A complementary DNA synthesis kit was purchased from Thermo Fisher Scientific (Waltham, MA). The forward and reverse primers used for reverse transcription polymerase chain reaction (RT-PCR) were synthesized by Macrogen (Seoul, Korea). PCR premix was purchased from Bio-D Inc. (Seoul, Korea). COX-1 and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA). Male ICR mice (age: 6–8 weeks; weight: 17–21 g) and male Sprague Dawley (SD) rats (age: 6 weeks; weight: 150-160 g) were purchased from Orient Bio (Gyeonggi, Korea). All other chemicals were obtained from Sigma Chemical Co.
5
Western Blot Analysis of Protein Expression
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Mice testes or cultured cells were homogenized in RIPA lysis buffer (Thermo Fisher Scientific, USA) containing protease inhibitor cocktail (Roche, USA) on ice for 30 min. Then centrifuged at 12000 g, 10 min, 4 °C. The proteins in the supernatant were collected and the protein concentrations were determined by the BCA Protein Assay Kit (Thermo Fisher Scientific).
Protein samples (20 μg) were separated by using 8–16% denaturing polyacrylamide gels, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) by using a semi-dry transfer apparatus (Bio-Rad, USA). Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and immunoblotting was performed overnight at 4 °C with the TFEB antibodies (1:5000 dilution; Santa cruz) or β-actin antibodies (1:4000 dilution; Cell Signaling Technology, USA), followed by incubation with secondary antibody conjugated to HRP (Jackson ImmunoResearch, USA). Signals were generated by enhanced chemiluminescence (Millipore) and detected by luminescent image analyzer (GE imagination LAS 4000, USA).
Protein samples (20 μg) were separated by using 8–16% denaturing polyacrylamide gels, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) by using a semi-dry transfer apparatus (Bio-Rad, USA). Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and immunoblotting was performed overnight at 4 °C with the TFEB antibodies (1:5000 dilution; Santa cruz) or β-actin antibodies (1:4000 dilution; Cell Signaling Technology, USA), followed by incubation with secondary antibody conjugated to HRP (Jackson ImmunoResearch, USA). Signals were generated by enhanced chemiluminescence (Millipore) and detected by luminescent image analyzer (GE imagination LAS 4000, USA).
6
Western Blot Analysis of Tryptophan Metabolism
7
Tamoxifen Modulates Inflammation and Kidney Injury
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Tamoxifen and lipopolysaccharide (Escherichia coli serotype 0111:B4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). TNF-α and IL-6 ELISA kits were obtained from Biolegend (San Diego, CA, USA). Blood urea nitrogen (BUN) assay kit was obtained from Arbor Assays (Ann Arbor, Michigan, USA). Kidney injury molecule-1 (KIM-1) assay kit was purchased from R&D (Minneapolis, MN, USA). Anti-Mouse Ly-6G (Gr-1)-FITC was purchased from eBioscience (San Diego, CA, USA). Goat anti-mouse ICAM-1 and VCAM-1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Phospho-STAT3 (Thr705), Stat3 (124H6), phospho-p44/42MAPK (ERK1/2) (Thr202/Tyr204), p44/42MAPK (ERK1/2), phosphorylated IκBα, IκBα, and β-actin antibodies were obtained from Cell Signal Technology (Boston, MA, USA).
8
Dihydroartemisinin-Nile Red Nanoparticle Synthesis
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Dihydroartemisinin (DHA) and Nile Red were bought from Aladdin (Shanghai, China). 2-nitroimidazole (2-MI) and indocyanine green (ICG) were purchased from J&K Scientific (Beijing, China). Zinc nitrate and bovine serum albumin (BSA) were purchased from Sigma–Aldrich (Merck Life Science, Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4, 6-diamidino-2-phenylindole (DAPI), and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were bought from Beyotime Biotechnology (Shanghai, China). DMEM medium and fetal bovine serum (FBS) were bought from Shanghai Titan Scientific Co., Ltd., (Shanghai, China). CAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), Bcl-2, and β-actin antibodies were bought from Cell Signaling Technology, Inc., (CST, Boston, MA, USA).
Cells were kindly provided by Pricella Life Science&Technology Co., Ltd., (Wuhan, China).
Cells were kindly provided by Pricella Life Science&Technology Co., Ltd., (Wuhan, China).
9
Latency Reversal Assay Protocol
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EIF2α, p-EIF2α (Ser51), RelA, NFATc1, c-fos, ATF3, HSF1, p-HSF1 (Ser320), p24, Lamin A/C, β-actin antibodies and secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Acetylated lysine antibody, p300, CDK9 and Cyclin T1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human FITC conjugated anti-CD25 and PE conjugated anti-CD69 antibodies were purchased from BD Biosciences (San Jose, CA, USA). PHA, poly I:C, STA-4783, thapsigargin, prostratin, PMA, ionomycin, SAHA, JQ1, dilazep, TNF-α, hemin, salubrinal, MG-132, BAY 11-7082, CsA, C646 and EX527 were from Sigma-Aldrich (St. Louis, MO, USA). Resveratrol, parthenolide (PTN), Ver-155008, KRIBB11, 17-DMAG and NVP-AUY922 were from Merck Calbiochem (Darmstadt, Germany). PEZ-HSF1 was purchased from ViGene Bioscience Inc (MD, USA). NL4-3E-R-luc plasmid, J-Lat 10.613 (link), U141 (link) and ACH242 (link) cell lines were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program.
J-Lat 10.6, U1 and ACH2 cell lines were maintained in RPMI1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) at 37 °C with 5% CO2. High temperature treatment (39.5 °C) was operated by putting cells in another incubator which temperature was adjusted to 39.5 °C.
J-Lat 10.6, U1 and ACH2 cell lines were maintained in RPMI1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) at 37 °C with 5% CO2. High temperature treatment (39.5 °C) was operated by putting cells in another incubator which temperature was adjusted to 39.5 °C.
10
Western Blot Analysis of Erythropoietin Signaling
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Total protein lysate (35 μg) from MGECs and MMECs were separated on 4–12% NuPAGE® gels (Invitrogen), electro-transferred to a polyvinylidene difluoride membrane (PerkinElmer Life Science Inc., Boston, MA) and immunoblotted with anti-Epo-R (M20, Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-pJAK2(Tyr1007/1008), anti-JAK2, anti-pSTAT5(Tyr694), anti-STAT5, anti-Akt, anti-pAkt(Ser473) (Cell Signaling Technology Inc., Danvers, MA) and β-actin antibodies (Sigma-Aldrich). Then, the membrane was incubated with mouse and rabbit horseradish peroxidase–conjugated IgG (Bio-Rad, Hercules, California, U.S.). Immunoreactive bands were visualized by enhanced chemiluminescence (LiteAblot extend substrate, Euroclone) and the Gel Logic 1,500 Imaging System (Eastman Kodak Co., Rochester, NY), quantified with the Kodak Molecular Imaging Software, and the expression bands were quantify as arbitrary optical density units (OD).
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