Supplemental Data Cell Metabolism, Volume 8 Inactivation of Hepatic Foxo1 by Insulin Signaling Is Required for Adaptive Nutrient Homeostasis and Endocrine Growth Regulation

Figure S1. Generation and supplementary characterization of floxed Irs1 and DKO-mice. (A) Scale map and scheme for generation of Irs1 floxed allele. Homologous recombination in ES cells between the linearized plasmid (i) and wild type chromosome (ii) yields the neomycin-resistant allele (iii) that, following mating of carriers of the allele to mice expressing Cre recombinase, results in the floxed Irs1 allele (iv). The positions of the loxP sites (lox) and splice donor and splice acceptor 3 ’ of the Irs1 coding region are shown, as are the approximate positions of probes ‘K ’ and ‘B ’ for identification of homologous recombinants by Southern blot of KpnI and BamHI-digested genomic DNA, respectively. Restriction sites in bold are unique to the plasmid construct. Restriction sites: K (KpnI), B (BamHI), N (NotI), Nh (NheI), X (XbaI). (B) Immunoblot analysis of Irs1 and Irs2 in skeletal muscle, liver, white adipose tissue (WAT), and brain of wild type and floxed Irs1 (Irs1L/L) mice. Alpha-tubulin was used as loading control. (C) Immunoblot analysis of tyrosine phosphorylated and total insulin receptors (IR) in the control floxed (CNTR), DKO and TKO-liver extracts after stimulation with insulin (5 units vena cava injected) (+) or control saline (-). (D) A longitudinal measurement of blood glucose i