PBMCs were lysed on ice for 30 min using RIPA lysis buffer (Beyotime) with protease inhibitors (Beyotime) and phosphatase inhibitors cocktail (Beyotime), and the protein concentration was determined by a bicinchoninic acid assay kit (Biosharp). The proteins (50 µg per lane) were mixed with one-fifth volume of 5× loading buffer, separated by 10% SDS-PAGE, and were then transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h with 5% nonfat milk at room temperature and were incubated overnight at 4°C with primary antibodies against STAT3 rabbit mAb (79D7, Cell Signaling Technology), phospho-STAT3-Tyr705 rabbit mAb (D3A7, Cell Signaling Technology), NF-κB p65 rabbit mAb (D14E12, Cell Signaling Technology), anti-NF-kB p65-phospho-S536 antibody (EP2294Y, Abcam), and β-actin rabbit monoclonal antibody (Beyotime). After 3 washes with Tris-buffered saline with 0.1% Tween® 20 detergent, the membranes were incubated for 1 h at room temperature with anti-rabbit horseradish peroxidase-conjugated secondary antibody (GE) and were detected with enhanced chemiluminescence (Beyotime). The results were analyzed using Image Studio software.
β actin rabbit monoclonal antibody
Manufactured by Beyotime
Sourced in China
The β-actin rabbit monoclonal antibody is a research-use only product that can be used to detect the β-actin protein, which is a commonly used loading control and housekeeping gene in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitors cocktail, by Beyotime (1 mentions)
Bicinchoninic acid assay kit, by Biosharp (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Enhanced chemiluminescence, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Phospho stat3 tyr705 rabbit mab d3a7, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by GE Healthcare (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Lysis extraction reagent, by Merck Group (1 mentions)
Anti human errα rabbit monoclonal antibody, by Abcam (1 mentions)
Enhanced chemiluminescence ecl detection system, by Thermo Fisher Scientific (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
P p38 mapk, by Cell Signaling Technology (1 mentions)
Calcein am pi double stain kit, by Beyotime (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Cell counting kit 8 (cck8), by Apexbio (1 mentions)
5 protocols using β actin rabbit monoclonal antibody
1
Western Blot Analysis of STAT3 and NF-κB Signaling
2
Western Blot Analysis of PPARγ and ERRα
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Cell lysates were prepared with a lysis/extraction reagent (Sigma). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay reagent kit (Pierce). Protein samples (30 μg) were resolved by 12% SDS-polyacrylamide gel electrophoresis, blotted onto PVDF membranes and blocked with TBS-Tween 0.1% containing 5% nonfat dry milk at room temperature for 1 h. Blotted membranes were incubated with anti-human PPARγ rabbit monoclonal antibody (1:500 dilution, Abcam, Cambridge, MA, USA) or anti-human ERRα rabbit monoclonal antibody (1:300 dilution, Abcam) overnight at 4°C. β-Actin rabbit monoclonal antibody (1:1000 dilution; Beyotime, Shanghai, China) was used as the control. An enhanced chemiluminescence (ECL) detection system (Thermo) was used to visualize the bands.
3
Western Blot Analysis of GR, NF-κB, and MAPK
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Cultured HNECs were seeded in a six-well plate, lysed, and then the protein was extracted by the RIPA lysis buffer combined with the protease and phosphatase inhibitor mix usage per well and quantified by the BCA kit usage. Proteins were separated using 10% polyacrylamide gel electrophoresis (PAGE). Every well was loaded with the protein sample and a protein marker on both sides, 5–10 µL, and 5 µL, respectively. After that, electrophoresis was done for 110 minutes at a 90 V constant voltage. The membrane was incubated with blocking buffer and incubated with GRα antibody ((4 μg.mL-1; Invitrogen, PA1516, USA), GRβ antibody (1:500; Invitrogen, PA3-514), p-P65 NF-κB (1:1000; Cell Signaling Technology, 3033T), p-P38 MAPK (1:1000; Cell Signaling Technology, 4511),
and β-actin rabbit monoclonal antibody (1:3000; Beyotime, AF5003, China) overnight at 4 °C. The membranes were treated with the HRP-labeled secondary goat anti-rabbit IgG antibody.
Immunoreactive proteins were measured using BeyoECL plus an Enhanced Chemiluminescence Western blot detection system.
and β-actin rabbit monoclonal antibody (1:3000; Beyotime, AF5003, China) overnight at 4 °C. The membranes were treated with the HRP-labeled secondary goat anti-rabbit IgG antibody.
Immunoreactive proteins were measured using BeyoECL plus an Enhanced Chemiluminescence Western blot detection system.
4
Cellular Stress Response Pathway Analysis
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Unless otherwise noted, solvents and reagents were obtained from commercial sources and used without further purification. Dulbecco’s modified eagle’s medium (DMEM, Gibco), phosphate buffered saline (PBS, Gibco), fetal bovine serum (FBS, Gibco), trypsin-EDTA (0.25%, Gibco), Alexa Fluor® 488-conjugated rabbit anti-phospho-histone H2A.X (Ser139) (20E3) mAb (Cell Signaling Technology, United States), Casepase 3 (active) rabbit monoclonal antibody (Beyotime Biotech, China), Bax rabbit monoclonal antibody (Beyotime Biotech, China), Bcl-2 rabbit monoclonal antibody (Sino Biological, China), β-actin rabbit monoclonal antibody (Beyotime Biotech, China), Calcein-AM/PI Double Stain Kit (Beyotime Biotech, China), Reactive Oxygen Species Assay Kit (Beyotime Biotech, China), Cell Counting Kit-8 (APExBIO Technology LLC, United States) were used as purchased.
5
Apoptosis and MAPK Pathway Analysis
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3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Beyotime. Annexin V-FITC apoptosis detection kit was obtained from Beyotime. The antibodies used in Western blot analysis, which included β-actin rabbit monoclonal antibody (1:1000), JNK1 + JNK2 + JNK3 rabbit monoclonal antibody (1:1000), phospho-JNK1/JNK2/JNK3 (Thr183/Thr183/Thr221) rabbit monoclonal antibody (1:1000), ERK1/2 rabbit monoclonal antibody (1:1000), phospho-Erk1 (Thr202/Tyr204)/Erk2 (Thr185/Tyr187) rabbit monoclonal antibody (1:1000), P38 MAPK rabbit monoclonal antibody (1:1000), phospho-P38 MAPK (Thr180/Tyr182) rabbit polyclonal antibody (1:1000), MMP2 rabbit polyclonal antibody (1:1000), MMP9 rabbit polyclonal antibody (1:1000), PARP1 rabbit polyclonal antibody (1:1000), Caspase-9 rabbit monoclonal antibody (1:1000), Caspase 3 rabbit polyclonal antibody (1:1000), Bax rabbit polyclonal antibody (1:1000), Bcl2 rabbit polyclonal antibody (1:1000), were obtained from Beyotime. Anti-rabbit (Beyotime) was used for the secondary antibody.
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