HeLa and N2a cells were harvested and lysed in Pierce RIPA buffer (ThermoFisher; #89901) with cOmplete proteinase inhibitor cocktail (Roche; #11697498001). Total protein levels were determined using a Bradford assay (ThermoFisher; #23236); Samples were diluted in 0.1× sample buffer (Bio-techne) and a total of 1.2 μg protein per sample was loaded for antibody staining. The primary antibody for human and mouse JAK1 was monoclonal anti-JAK1 (Cell Signaling; #50996) in a 1:500 dilution; and for human and mouse housekeeping β-actin was monoclonal anti-β-actin (Cell Signaling; #4970) in a 1:500 dilution. Secondary antibodies used were: Anti-mouse detection module (Bio-techne; #DM-002) and anti-rabbit detection module (Bio-techne; #DM-001). The assay was performed as described by the ProteinSimple protocol using the 16–230 kDa separation module (Bio-techne; SM-W004) on a Wes system (Bio-techne). For in vivo studies, footpad skin biopsies were mechanically homogenized in 500 μL Pierce RIPA buffer proteinase inhibitor cocktail, the processed skin homogenate was centrifuged at 10,000×g for 5 min, and the clear supernatant was collected for Wes ProteinSimple assay. The primary antibody for mouse JAK1 was monoclonal anti-JAK1 (Cell Signaling; #50996) in a 1:500 dilution; and for mouse housekeeping β-actin was monoclonal anti-β-actin (Cell Signaling; #4970) in a 1:250 dilution.
Monoclonal anti β actin
Manufactured by Cell Signaling Technology
Sourced in United States
Monoclonal anti-β-actin is a laboratory reagent used for the detection and quantification of β-actin, a highly conserved cytoskeletal protein, in various biological samples. This antibody is a useful tool for monitoring protein expression levels in cellular and tissue-based studies.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti smad5 monoclonal, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Immobilon p, by Merck Group (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Monoclonal anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Monoclonal anti bcl 2, by Cell Signaling Technology (1 mentions)
Hrp conjugated immunoglobulin, by Jackson ImmunoResearch (1 mentions)
Image master vds, by GE Healthcare (1 mentions)
Calponin, by Boster Bio (1 mentions)
Osteopontin opn, by Boster Bio (1 mentions)
Anti p stat1, by Cell Signaling Technology (1 mentions)
Anti inos, by Cell Signaling Technology (1 mentions)
Anti cox 2, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Curcumin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Cell Signaling Technology (1 mentions)
Nitrocellulose, by Bio-Rad (1 mentions)
14 protocols using monoclonal anti β actin
1
Quantitative Western Blot Protein Assay
2
Quantification of ADRB2 Protein Expression
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A radioimmunoprecipitation assay lysis buffer (Upstate Biotechnology, Inc., Lake Placid, NY, USA) with protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) was utilized to lyse the cells 48 h post-transfection to detect the expression level of ADRB2 protein, in accordance with the manufacturer's protocol. The protein was quantified using the bicinchoninic acid method. SDS gradient gels of 4–20% (Mini-PROTEAN TGX; Bio-Rad Laboratories, Inc., Hercules, CA, USA) were utilized to load equal amounts (30 µg) of total protein, and gel electrophoresis was performed to separate the proteins, followed by transfer to polyvinylidene fluoride membranes (Immobilon-P; EMD Millipore, Billerica, MA, USA). The primary antibodies anti-ADRB2 at a dilution of 1:5,000 (cat. no. 8513; Cell Signaling Technology, Inc., Beverly, MA, USA) and monoclonal anti-β-actin at a dilution of 1:10,000 (cat. no. sc-130301; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were utilized to treat the membrane at 4°C overnight, and horseradish peroxidase (HRP)-conjugated secondary anti-rabbit immunoglobulin G antibody at a dilution of 1:10,000 (cat. no. ab6721; Abcam, Cambridge, MA, USA) was used to treat the membrane for 60 min at room temperature. ImageJ software version 1.0 (National Institutes of Health, Bethesda, MD, USA) was used to visualize the bands. Each test was repeated in triplicate.
3
Western Blot Analysis of Apoptosis and Signaling
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Cell lysates were prepared and 20 µg of these were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Specific monoclonal anti-cleaved caspase-3 [Cell Signal Technology (CST), SN: 4380, dilution: 1:2000], monoclonal anti-Bcl-2 (CST, SN: 11988, dilution: 1:2000), monoclonal anti-p65 (CST, SN: 5741, dilution: 1:2000), monoclonal anti-p-p38 (CST, SN: 3195, dilution: 1:2000), and monoclonal anti-β-actin (CST, SN: 8457, dilution: 1:4000) antibodies were used. HRP-conjugated immunoglobulin was used as the secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). West Pico chemiluminescence was used as the substrate to visualize protein bands, which were quantified using densitometric image analysis software (Image Master VDS; Pharmacia Biotech) and normalized to β-actin expression.
4
Western Blot Analysis of Aortic Vascular Media
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The vascular media of thoracic aorta tissue of every rat was homogenized in lysis buffer and centrifuged at 14000 × g for 15 minutes at 4°C, then the supernatant was collected. Electrophoresis was performed on 10% SDS-polyacrylamide gels to transfer proteins to 0.45 mm Sequi-Blot PVDF membranes (23 V, 55 minutes). After incubation with 5% non-fat milk, then incubated overnight at 4°C with primary antibodies against CaSR (1:800, Abcam, Massachusetts, USA), proliferating cell nuclear antigen (PCNA, 1:400, Boster, Wuhan, China), smooth muscle a-actin (SMAa) (1:400, Boster, Wuhan, China), calponin (1:200, Boster, Wuhan, China), osteopontin (OPN) (1:200, Boster, Wuhan, China). Monoclonal anti-β-actin (1:1000, Cell Signaling Technology, Danvers, MA, USA) antibodies were used to standardize each line and as an integral loading control. After being washed, the membranes were incubated with horseradish peroxides (HRP)-conjugated species-specific secondary antibodies (1:25000, Boster, Wuhan, China). Protein was visualized using an enhanced chemiluminescence system (ECL, Pierce Company, USA). Intensities of the protein bands were quantified using a Bio-Rad Quantity One software (Bio-Rad, Hercules, CA, USA).
5
Quantifying Inflammation Markers
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Monoclonal anti-collagen I, anti-TIMP-1 and anti-matrix metalloprotease-1(MMP-1) were obtained from Signalway (Pirland, Texas, USA), and monoclonal anti-plasminogen activator inhibitor 1(PAI-1) was from KeyGEN Ltd., (Nanjing, Jiangsu, China). Monoclonal anti-β-actin, anti-p-STAT1, anti-COX-2 and anti-iNOS were purchased from Cell Signaling Technology (Danfoss, Massachusetts, USA).
6
Curcumin Modulates BC Cell Lines
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The T24 and RT4 human BC cell lines were obtained from the Chinese Academy of Science (Shanghai, China). The BC cells were cultured in Dulbecco's modified Eagle's medium (DMEM; cat. no. MGC803; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% FBS and 100 U/ml penicillin/strep tomycin (HyClone™; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified atmosphere containing 5% CO2. Curcumin (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) was dissolved in dimethylsulfoxide (DMSO) and stored at −20°C. 3–4,5-dimethyl-2- thiazolyl-2, 5-diphenyl-2-H-tetrazolium bromide (MTT; CAS no. 57360-69-7) was obtained from Sigma-Aldrich; EMD Millipore. Lipofectamine 2000 was purchased from Invitrogen; Thermo Fisher Scientific, Inc. Primary antibodies targeting Trop2 (#90540), p27 (#2552), and cyclin E1 (#4129), and monoclonal anti-β-actin (#3700) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies (#A-11031 and #A-11034) were obtained from Thermo Fisher Scientific, Inc.
7
Protein Expression Profiling of CD34+ Cells
8
Western Blot Analysis of Inflammatory Markers
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The hearts, lungs and livers of rats were isolated 6 h after LPS initiation and were immediately frozen in liquid nitrogen and stored at -80°C until processed. Detection of protein expression by Western blotting was performed as described previously [21 (link)]. The primary antibodies used in this experiment were mouse monoclonal anti-iNOS (1:1000; BD, USA), monoclonal anti-HO-1 (1:2000; Santa Cruz, USA), monoclonal anti-HSP70 (1:1000; Assay Designs, USA), monoclonal anti-plasminogen activator inhibitor-1 (PAI-1) (1:10000; BD, USA), monoclonal anti-β-actin (1:5000; Cell Signaling, USA), monoclonal anti-α-actin (1:2000; Sigma, USA) and rabbit polyclonal phosphorylated NF-κB p65 (pp65) antibody (1:2000; Millipore, USA).
9
Retinal Protein Expression Analysis
10
Quantifying Phospho-eNOS Expression in EPCs
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Phospho-eNOS expression in EPCs was measured by western blot. Total proteins were obtained from cells using cell lysis buffer and were quantified with BCA Protein Assay Kit (Thermo Scientific). 50 μg of proteins were subjected to 8% SDS-PAGE and then transferred onto a PVDF membrane. Primary antibodies against anti-phospho-eNOS (Ser 1177) monoclonal (1: 1000, EnoGene) and anti-β-actin monoclonal (1: 1000, Cell Signaling Technology) were incubated at 4°C overnight. Next, the membranes were blotted with the horseradish peroxidase-conjugated anti-rabbit IgG for 1 h (1: 3000, Beijing Zhongshan Golden Bridge Biotechnology). The membranes were washed and proteins were detected using a Western Blotting Luminol Reagent systerm and autoradiography.
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