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6 protocols using anti pcna

1

Western Blot Analysis of Cell Signaling

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Cells were lysed with radio immunoprecipitation assay buffer (RIPA) containing 1 mmol/L PMSF (Solarbio, China). Protein extractions were separated by 12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Germany). The membranes were incubated overnight at 4 °C with primary antibodies specific for anti-β-actin, anti-CDK2, anti-CyclinD1, anti-CyclinE, anti-PCNA, and anti-P53 (1:1,000; Wanleibio), anti-Bcl-2, anti-Bax, anti-Caspase9, anti-FOXC1, and anti-MyoD1 (1:1,000; Abways Technology), anti-MyoG, (1:1,000; Abcam, Cambridge, England). After incubation with secondary antibodies, signals were detected using a chemiluminescence system (Bio-Rad, USA).
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2

Western Blot Analysis of Bovine Adipocytes

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Total proteins of bovine fat cells were extracted using radioimmunoprecipitation assay buffer (RIPA) buffer with 1% PMSF (Solarbio, Beijing, China) after transfection for 24 h or induction for 6 days after transfection. The protein concentration was determined by a bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China), a 5× protein loading buffer (containing mercaptoethanol) was added to proteins at a ratio of 1:4, and then the sample was heated in boiling water for 3–5 min. Proteins were then separated by SDS-polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes and blocked with milk powder solution for 2 h at room temperature. The membranes were then incubated overnight with the primary antibody of anti-CDK2, anti-PCNA, anti-PPARγ, anti-C/EBPβ, and anti-β-actin (Wanlei Bio, Shenyang, China). The membranes were then washed with PBS-Tween 20 and incubated for 1.5 h with horseradish peroxidase-conjugated secondary antibodies (Boster, Pleasanton, CA, USA). Finally, the membranes were imaged with an enhanced chemiluminescence (ECL) kit (Solarbio, Beijing, China) and quantified with the ImageJ program (Bio-Rad, USA).
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3

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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4

Immunohistochemistry Staining Protocol for Tissue Analysis

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IHC was performed according to standard procedures. The paraffin sections of xenograft were randomly selected to detect all biomarkers. For clinical FFPE tissue specimens, the tissue slices were randomly selected from the same one to detect all biomarkers. The primary antibodies anti‐NR6A1 (1:50, Proteintech, China, 12712‐1‐AP), anti‐MAP2(1:8000, Abcam, ab1838830), anti‐PCNA (1:100, WanleiBio, China, WL03213,), anti‐E‐cadherin (1: 200, CST, #3195), and DAB (ZSGB‐Bio, China) were used for staining. The results were obtained by digital slice scanner (3DHISTECH, Hungary). The staining score of IHC was estimated as negative (0), weak (1), moderate (2), and strong (3). The extent of staining, defined as the percentage of positive stained cells, was scored as 1 (≤10%), 2 (11%−50%), 3 (51%−80%), and 4 (> 80%). The total immune reactive score (IRS) was obtained by multiplying the score of intensity and that of extent, ranking from negative (−) to >6 (+++).26
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5

Comparative Protein Expression Analysis of Myoblasts and Preadipocytes

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Proteins from cultured myoblasts and preadipocytes were lysed with RIPA buffer (Solarbio, Beijing, China). Proteins were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene di uoride (PVDF) membranes (Thermo Fisher Scienti c). The membranes were incubated overnight with primary antibodies speci c for anti-GAPDH (#ab9485), anti-CyclinD1 (#ab226977), (Abcam, Cambridge, UK), anti-Bcl-2 (#bs-0032R), anti-caspase-9 (#bs-0049R), anti-Bax (#bs-0127M), anti-FABP4 (#bsm-51247M) (Bioss, Beijing, China), anti-PCNA (#WL01804), anti-CDK2 (#WL01543), anti-PPARγ (#WL01800) and anti-C/EBPα (#WL01899) (Wanlei Bio, Shenyang, China) at 4℃. After incubation with secondary antibodies, the membranes were quanti ed with the chemiluminescence system (Bio Rad, Hercules, CA, USA).
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6

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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