Anti igf1rα

Primary antibodies used in this study were purchased from Cell Signaling Technology (Beverly, MA): anti-pPKA^Thr198/PKA, anti-pCREB^Ser133/CREB, anti-pSTAT3^Tyr705/STAT3, anti-pSrc^Ser17/Src, anti-pACC ^Ser79/ACC, anti-pAkt^Ser473/Akt, anti-pAMPK ^Thr172/AMPK, anti-p4EBP1^Thr37/46/4EBP1, anti-pmTOR^Ser2448/mTOR, anti-pP70S6K^Thr389/P70S6K, anti-EPAC-1, anti-BEATA2_AR, anti-FAK, anti-FASN, anti-GLUT4, anti-OCT3, anti-NOTCH, anti-PGC1, anti-pPRAS40^Thr246, anti-PRAS40, anti-pRAPTOR^Ser792, 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).

All samples were analyzed by SDS-PAGE on 8% acrylamide gels and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore). Primary antibodies used were anti-SBP-tag (Millipore, MAB10764, Clone 20, 1:1000 dilution), Anti-IRα (Santa Cruz, sc-7953, H-78, 1:500 dilution), Anti-IRβ (Cell Signaling, #3025, 4B8, 1:1000 dilution), Anti-IGF1Rα (Santa Cruz, sc-271606, G-5, 1:1000 dilution), Anti-IGF1Rβ (Cell Signaling, #14534, D4O6W, 1:1000 dilution), Anti-pYpY (IR phosphorylated Tyr^1162/1163 and IGF1R phosphorylated Tyr^1135/1136, Calbiochem, 407707-10 T, 1:1000 dilution). Secondary antibodies used were horseradish peroxidase conjugated antibodies of either rabbit IgG (GE Healthcare Life Sciences,1:10,000 dilution) or mouse IgG (GE Healthcare Life Sciences, 1:10,000 dilution). Lastly, membranes were incubated with Western blotting substrate (Thermo Scientific Pierce ECL) and exposed to autoradiographic film (BioExcell).