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2 protocols using anti pi 3 kinase p85α

1

Western Blot Analysis of Signaling Proteins

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Cells were cultured or treated by rBFT1, followed by harvesting at predetermined time points. After extensive washing, cells were lysed using Whole Cell Lysis Assay (KeyGEN Biotech, China) according to the manufacturer’s instructions and proteins from the lyste were harvested by centrifugation at 12,000 × g for 5 min at 4°C. Protein concentrations were determined by the BCA Assay kit (KeyGEN Biotech, China). Total protein (42 kDa) from each lysate was then separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, MA, USA). Afterwards, the membranes were blocked with 5% non-fat milk in TBST at room temperature for 1 h, followed by incubation with antibodies against protein of interest overnight at 4°C. The antibodies used in this study included anti-NF-κB/P65 (1:100 in PBS), anti-Macrophage Inflammatory Protein 1α/CCL3 (100 μg/mL in PBS), anti-CCR5 (1:100 in PBS), anti-PI 3 Kinase/P85α (1:200 in PBS), and anti-TRAF6 (1 µg/mL in PBS) (Abcam, CA, USA). After washing with TBST, membranes were striped with appropriate HRP-conjugated secondary antibodies (1:5,000, Abcam, CA, USA) for 1 h at room temperature, followed by visualization using the enhanced Western Bright ECL reagents (Cell Signaling Technology, USA). Protein bands were scanned and analyzed by a chemiluminescence detection system (Bio-Rad, USA).
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2

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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