Cells (3×106/dish) were seeded into 100-mm2 tissue culture dishes and treated with individual cranberry flavonoids. Cells were then lysed with cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) supplemented with 5 μl/ml phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations of cell lysates were determined with Pierce BCA protein assay kit (Pierce Technology, Rockford, IL, USA). Gel electrophoresis was performed in NuPAGE Gel system (Life Technologies) according to the manufacturer’s instructions. Separated proteins were transferred onto a nitrocellulose membrane, which was then blocked with 5% non-fat milk in PBS-Tween buffer and probed against various primary antibodies (cleaved caspase-3 no. 9664, cleaved PARP no. 5625, EGFR no. 4267, phospho-EGFR no. 3777, phospho-c-Raf no. 9427, phospho-ERK1/2 no. 4370, phospho-p53 no. 9286, p18 INK4C no. 2896, p21 Waf1/Cip1 no. 2947, p27 Kip1 no. 3686, CDK2 no. 2546, cyclin D1 no. 2926, cyclin D3 no. 2936, β-actin no. 8457, β-tubulin no. 2128; Cell Signaling Technology). Protein bands were visualized using horseradish peroxidase conjugated anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology) and Pierce ECL Western Blotting Substrate, and documented by Bio-Rad Gel Doc system (Bio-Rad, Hercules, CA, USA).
β actin no 8457
Manufactured by Cell Signaling Technology
β-actin antibody no. 8457 is a primary antibody that detects the β-actin protein. β-actin is a ubiquitously expressed cytoskeletal protein that plays a crucial role in maintaining cell structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc system, by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate, by Bio-Rad (1 mentions)
Cdk2 no 2546, by Cell Signaling Technology (1 mentions)
Phospho c raf, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Phycoerythrin pe, by BioLegend (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
2 protocols using β actin no 8457
1
Cranberry Flavonoid Effects on Cell Signaling
2
Antibody Panel for Immune Phenotyping
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Phycoerythrin (PE)-, PE/Cy7, Alexa 647, allophycocyanin (APC)-, or APC/Cy7-conjugated anti-mouse CD4 (GK1.5), CD8 (53-6.7), CD25 (3C7), CD45RB (C363-16A), CD25 (3C7), CD44 (IM7), CD117 (2B8), TCRVβ (H57-597), TGF-β1 (TW7-16B4), FoxP3 (MF-14), and acetylated lysine (15G10; no. 623402) were purchased from BioLegend (San Diego, CA). Rabbit NAC1 (no. 4183), HDAC6 (no. 7612), and β-actin (no. 8457) antibodies were purchased from Cell Signaling (Beverly, MA). Rabbit anti-NAC1 antibody (ab29047) for immunoprecipitation was obtained from Abcam (Cambridge, MA). Mouse HDAC9 (no. sc-398003) and HDAC11 (no. sc-390737) antibodies were purchased from Santa Cruz Biotechnologies (Dallas, TX). Cycloheximide were purchased from Sigma-Aldrich Corporation (Sigma-Aldrich, St. Louis, MI).
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