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3 protocols using anti p21

1

Western Blot Protein Expression Analysis

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Cells were lysed in RIPA lysis buffer (BD Pharmingen, San Diego, CA, USA), protein concentration was measured by BCA reagent kit (Beyotime Institute of Biotechnology, Haimen, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate cellular lysates, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA, USA). The transfected membranes were blocked in 5% skim milk and incubated in 4°C overnight using the following primary antibodies: anti-ATF1 (1:1,000), anti-ATM (1:1,000), anti-ATM (phospho-1981; 1:5,000) (all from Abcam, Cambridge, MA, USA), anti-p53 (1:800), anti-p21 (1:500), anti-Bax (1:1,000) and anti-Bcl-2 (1:500) (all from Wanleibio, Shenyang, China), anti-GAPDH (1:3,000; Proteintech Group, Inc., Chicago, IL, USA). Then incubated with horseradish peroxidase-conjugated secondary antibody, goat anti-rabbit (1:5,000) or goat anti-mouse (1:5,000) (both from Cell Signaling Technology, Inc., Danvers, MA, USA). All bands were visualized with enhanced chemiluminescence (ECL) kit (Millipore).
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2

Protein Expression Profiling in Bovine Myoblasts

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Total proteins of bovine myoblasts were prepared with radio immunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China). Proteins were fractionated on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). Blots were incubated overnight with primary antibodies specific for anti-CyclinD1 (#ab226977), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #ab9485), anti-MyoD (#ab16148) (Abcam, Cambridge, UK), anti-INSR (#WL02857), anti-PCNA (#WL01804), anti-CDK2 (#WL01543), anti-CyclinE (#WL01072), anti-Bcl-2 (#WL01556), anti-P53 (#WL01919), anti-P21 (#WL0362), anti-MyoG (#WL01132), anti-MyHC (#WL02785), and anti-Bax (#WL01637) (Wanleibio, Haerbin, China) at 4°C. After incubation with secondary antibodies, the membranes were quantified with the ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).
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3

Curcumol Inhibits Cancer Cell Growth

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Curcumol (purity > 98%) was purchased from Guizhou Dida Technology Co., Ltd (Guizhou, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Amresco, LLC (Solon, OH, USA). DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Anti-rabbit IgG, anti-mouse IgG and antibodies against β-actin were purchased from ZSGB-BIO (Beijing, China). The anti-PI3 Kinase P110 β, anti-PI3 Kinase P85 α, anti-Nucleolin, anti-AKT, anti-phosphor(p)-AKT, anti-PARP-1, anti-P53, anti-Bcl-2 were purchased from Abcam (Cambridge, MA, USA), anti-CDK6, anti-CyclinD1, anti-CDK2 were purchased from Cell Signaling Technology (Danvers, MA, USA), anti-PCNA, anti-P21 were purchased from Wanleibio, LLC (Shenyang, China), anti-ERα36 antibody and plasmid vector were provided by Zhao Yi Wang (Creighton University Medical School).
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