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2 protocols using camp assay kit

1

Western Blot Analysis of Signaling Proteins

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Primary antibodies used in this study were purchased from Cell Signaling Technology (Beverly, MA): anti-pPKAThr198/PKA, anti-pCREBSer133/CREB, anti-pSTAT3Tyr705/STAT3, anti-pSrcSer17/Src, anti-pACC Ser79/ACC, anti-pAktSer473/Akt, anti-pAMPK Thr172/AMPK, anti-p4EBP1Thr37/46/4EBP1, anti-pmTORSer2448/mTOR, anti-pP70S6KThr389/P70S6K, anti-EPAC-1, anti-BEATA2_AR, anti-FAK, anti-FASN, anti-GLUT4, anti-OCT3, anti-NOTCH, anti-PGC1, anti-pPRAS40Thr246, anti-PRAS40, anti-pRAPTORSer792, anti-RAPTOR, and anti-PI3K110α. Anti-BCL-2 was purchased from BD Bioscience (San Jose, CA). Anti-P27 was purchased from Thermo Fisher Scientific (Waltham, MA). Anti-rabbit immunoglobulin-horseradish peroxidase-conjugated secondary antibody, LumiGLO reagent with peroxide and cAMP assay kit were also purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-IGF1Rα, anti-HMGCR, anti-P21, anti-SCD1, and anti-SCEBP1 were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX); mouse anti-β-actin primary antibody was obtained from Sigma Aldrich (St. Louis, MO). The 1-methyl-1-nitrosourea (MNU) was obtained from Ash Stevens (Detroit, MI) and stored at −80°C prior to use. Metformin and buformin were obtained from Waco Pure Chemical Industries, (Waco, TX); phenformin was obtained from Sigma Aldrich (St. Louis, MO).
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2

Epididymal Adipose cAMP Measurement

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Epididymal adipose tissue supernatants were obtained by homogenate and centrifugation. Cyclic AMP in the supernatants were measured using cAMP assay kit (4339, Cell Signaling Technology) according to the manufacturer’s protocol.
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