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2 protocols using anti cyclin d3

1

Cell Viability and Protein Expression Assay

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Cell culture media, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hematoxylin and eosin were purchased from Sigma. Foetal bovine serum (FBS) and antibiotics were from Life Technologies and Immobilon P (PVDF) membranes from Millipore. OOS was provided by Catalysis, S.L. (Madrid, Spain). The Cytometric Bead Array Mouse Inflammation kit was purchased to BD biosciences. Other generic chemicals were purchased from Sigma Chemical Co., Roche Biochemicals or Merck.
The origin of the different antibodies used in the Western blotting analyses were as follows: the anti-GAPDH, anti-p21, anti-Wee1, anti-CDC2/CDK1, anti-pCDC2, anti-Cyclin B and anti-Cyclin D1 antibodies were purchased from Santa Cruz Biotechnology; the anti-BUBR1, anti-Rb, anti-Cyclin A, anti-Cyclin D3 and anti-Cyclin E from BD Biosciences; the anti-p27 and the anti-pRbS780 from Cell Signaling technology; the anti-Calnexin from Stressgen and the anti-pHistone H3 from Millipore. The horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad.
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2

Viability and Immunoblot Assay Protocol

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For the viability assay, the CellTiter-Glo 2.0 Assay kit was purchased from Promega (Cat #G9243, Madison, WI). For immunoblotting, the following antibodies were purchased: anti-rat NF-κB p65 (dilution– 1:750, Thermo Fisher, Cat #436700, Waltham, MA), anti-TNF-α (dilution– 1:1000, Thermo Fisher, Cat #BMS175, Waltham, MA), anti-AKAP95 (dilution– 1:2500, gifted by Vince Coghlan, OHSU, Portland, OR), anti-cyclin D3 (dilution– 1:1000, BD Biosciences, Cat #610279, Franklin Lakes, NJ), and anti-actin (dilution– 1:1000, Sigma-Aldrich, Cat #A2066, St. Louis, MO). For TNF ELISA, the Rat TNF-alpha ELISA kit was purchased from RayBiotech (Cat #ELR-TNFa-1, Norcross, GA).
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