For the Western blot analysis, cells were harvested and lysed in protein lysis buffer (Merck Millipore, St. Louis, MO, USA) supplemented with a complete protease inhibitor cocktail. Cell lysates were prepared in a previously described manner [40 (link)]. Primary antibodies against PAR1 (ATAP2:sc-13503; Santa Cruz Biotechnology, Dallas, TX, USA), RhoA, ROCK1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling, Danvers, MA, USA), and β-actin were purchased from Novus Biologicals (Littleton, CO, USA), both GAPDH and β-actin were used as loading controls. Appropriate HRP-conjugated secondary antibodies were used to detect proteins using a luminol-based enhanced chemiluminescent (ECL) substrate (Thermo Scientific). An ImageQuant LAS 4000 analyzer (GE Healthcare Life Science, Pittsburgh, PA, USA) was used to detect protein expression levels.
β actin
Manufactured by Novus Biologicals
Sourced in United States, China
β-actin is a highly conserved cytoskeletal protein that is essential for maintaining the structural integrity of cells. It is a key component of the actin cytoskeleton and plays a critical role in various cellular processes, including cell motility, cell division, and intracellular transport.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (6 mentions)
Caspase 3, by Cell Signaling Technology (5 mentions)
P38 mapk, by Cell Signaling Technology (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Gapdh, by Novus Biologicals (3 mentions)
Phospho p38 mapk, by Cell Signaling Technology (3 mentions)
Phospho akt, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
P62 sqstm1, by Abnova (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pgsk 3β ser9, by Novus Biologicals (2 mentions)
Gsk 3β, by Novus Biologicals (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Phosstop, by Roche (2 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
101 protocols using β actin
1
Western Blot Analysis of PAR1, RhoA, ROCK1
2
Comprehensive Protein Analysis Protocol
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Cells were lysed in SDS lysis buffer (240 mM Tris-acetate, 1% SDS, 1% glycerol, 5 mM EDTA pH 8.0) with DTT, protease inhibitors, and a cocktail of phosphatase inhibitors. Antibody against vimentin, and MCP-1 were purchased from Abcam (Cambridge, MA, USA). Antibodies detecting CD44 and phospho-YAP S127 were purchased from GeneTex, Inc. (Irvine, CA, USA). Antibodies against YAP, phospho-MST1/MST2 T183/T180, MST1, phospho-LATS1 S909, LATS1, CYR61, CTGF, phospho–AKT T308, AKT, and phospho–GSK3β S9, phospho-ERK1/2, ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). The β-actin, GAPDH, and α -tubulin antibodies were purchased from Novus Biologicals (Littleton, CO, USA). Antibody against E-cadherin was purchased from BD (Franklin Lakes, NJ, USA).
3
Western Blot Analysis of Immune Markers
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Cell lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes. Membranes were incubated with antibodies against IDO (Millipore, Bedford, MA), TGF-β, STAT3, phospho-STAT3, CCL2/MCP-1 (Cell Signaling, Beverly, MA), IL-10, VEGF (Abcam, Cambridge, MA), and β-actin (Novus, Littleton, CO) at 4°C overnight after blocked with 5% nonfat milk. After washing with Tris-Tween buffer saline, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 1 hour. The proteins of interest were visualized by the luminescence imaging system LAS-4000 (Fujifilm, Tokyo, Japan) with the ECL chemiluminescent detection system (Millipore). β-actin was used as an internal control, and all the proteins were normalized to their own β-actin before compared with that of the control. ImageJ (National Institutes of Health, Bethesda, MD) was used for the quantitative analysis. The experiments were repeated more than three times.
4
Subcellular Protein Extraction and Western Blotting for ER Stress Markers
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NRK52E cells were treated with 30 µM NF as indicated. The extraction of cytoplasmic and nuclear proteins was carried out as instructed by the cytoplasmic and nuclear protein extraction kits (Biotools, New Taipei City, Taiwan). The proteins obtained were transferred to new Eppendorf flasks and stored at −80 °C. A standard protocol for Western blotting was used as described previously [35 (link)]. The specific primary antibodies used in this study included IRE1a, p-IRE1a, β-actin (Novus Biologicals, Littleton, CO, USA), PERK, p-PERK (Bioss Antibodies, Woburn, MA, USA), ATF6α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), GRP78 (Epitomics, Abcam, Cambridge, MA, USA), CHOP, caspase-7, caspase-3 (Cell Signaling, Danvers, MA, USA), and caspase-12 (BioVision, Milpitas, CA, USA). Western blotting was repeated at least three times.
5
Western Blot Protein Quantification
6
Western Blot Analysis of Liver Proteins
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Pieces from the liver were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitors. The protein concentration in the homogenates was determined using Bradford reagent [39 (link)] and 40 µg proteins were subjected to 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The separated proteins were transferred to nitrocellulose membranes which were blocked using 5% skimmed milk in tris buffered saline/tween 20 (TBST). After blocking, the membranes were probed with antibodies against pAkt Ser473, Akt, pGSK-3β Ser9, GSK-3β, and β-actin (Novus Biologicals, Centennial, CO, USA) overnight at 4 °C. The blots were washed three times with TBST and incubated with the secondary antibodies for 1 h at room temperature. The membranes were washed three times with TBST and developed using enhanced chemiluminescence detection kit (BIO-RAD, Hercules, CA, USA). The developed blots were scanned, and the band intensity was quantified using ImageJ (version 1.32j, NIH, USA).
7
Western Blot Analysis of Cell Cycle Regulators
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Cells were lysed with RIPA buffer to extract proteins. A total of 10 μg of each protein extract was electrophoresed on a sodium dodecyl sulfate-(SDS)-polyacrylamide gel, and then the resolved proteins were transferred to a nitrocellulose membrane. Blocked membranes were incubated with primary antibodies against phospho-p53 (pho-p53), p16, phospho-Rb (pho-Rb), p21, p53, Rb (Cell Signaling), and p16 (Abcam, Cambridge, UK), followed by horseradish peroxidase-conjugated secondary antibodies. Chemiluminescent intensity of immunoblotted bands was visualized using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). The intensity of each band was normalized to that of β-actin (Novus Biologicals, Centennial, CO, USA).
8
Mouse Immune Cell Isolation and Signaling
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DMEM-F12, Advanced DMEM, DPBS, FBS, TrypLETM Express, pen-strep, glutamax, and protease inhibitor were purchased from Gibco Life Technologies (Grand Island, NY, USA). TGF-β and ROCK inhibitors were procured from Tocris Bioscience (Bristol, UK). Mouse rIL-20 was procured from BioNovus Life Sciences (NSW, Australia). All phosphorylated antibodies against STAT1, STAT3, STAT5, Akt, 42/44MAPK (ERK1/2), NF-κBp65, 90RSK, and c-Jun, secondary antibodies against rabbit and mouse, were obtained from Cell Signaling Technology (Danvers, MA, USA) and β-actin from Novus Biologicals (Littleton, CO, USA). Phosphatase inhibitor PhosSTOP was purchased from Roche (Basel, Switzerland). Antibodies against mouse CD3, CD45, and F4/80 were procured from BD Bioscience (CA, USA), where mouse specific CD4, CD11b, CD11c, CD44, α4β7 were found, and Zombie GreenTM fixable viability kit were from Biolegend (CA, USA).
9
Western Blot Analysis of Cellular Proteins
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Protein lysates were isolated from cells and run on 4 to 15% Tris⋅HCl poly-acrylamide gels. Proteins were transferred to nitrocellulose membranes and blocked with 1:1 Intercept PBS Blocking Buffer (LI-COR):PBS. Membranes were incubated in the following primary antibodies: HIF2α (Novus Biologicals NB100-122), VHL (Novus Biologicals, NB100-485), TET-2 (Cell Signaling Technology, 18950), TET-1 (Genetex, GTX124207), TET-3 (Genetex, GTX121453), HIF1α (Novus, NB100-105), SDHD (Abcam, ab189945), SDHB (Abcam, ab14714), Lamin A (Cell Signaling, 133A2), α-Tubulin (Cell Signaling, 2144S), and β-Actin (Novus Biologicals, NB600-501; AC-15). Proteins of interest were detected using IRDye 800CW and 680RD secondary antibodies (LI-COR) on a LI-COR Odyssey Fc Imaging System.
10
Antibody Detection for Autophagy Signaling
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