Analysis of hypothalamic function in the fIrs2:cr² mice. (A) Immunoblots of hypothalamus lysates. After an overnight fasting, anesthetized 8-week-old mice were injected intravenously with 50 μl of saline or saline containing 5 units of insulin. Fifteen minutes later, the hypothalamus was isolated and lysed. Lysates were immunoprecipitated with anti_Irs2 antibody, resolved by SDS-PAGE, and then immunoblotted with antibodies against Irs2, phosphotyrosine (pY20), or p85. Lysates were also blotted independently with antibodies against Akt or phospho-Akt [Akt(Pi)], or Stat3. Data are representative of at least three independent experiments. (B) The relative expression of various mRNAs_Npy (1421690_s_at), Agrp (1419127_at), Cart (1422825_at) or Pomc1 (1455858_x_at; 1433800_a_at), Stat3 (1426587_a_at; 1426587_a_at; 1460700_at), Socs3 (1455899_x_at; 1456212_x_at) or PcSK2 (1448312_at; 1444147_at) was determined in hypothalamic extracts using Affymetrix GeneChip Mouse Expression Array 430A. The fold change is shown with the 90% confidence interval for expression in 3 fIrs2:cr² mice compared with the average control values from two WT, 2 cr² and 2 fIrs2 mice. Where more than one probe occurs on the Genechip, the average is reported. Cntr, control. (C) Brain sections from WT or fIrs2:cr² mice were immunostained with antibodies against αMSH (green) or Irs2 (red). Yellow appears in the digitally merged images where αMSH and Irs2 are coexpressed. Magnification, ×20; *3rd ventricle. (D) Body weight and blood glucose levels of fIrs2:cr², [fIrs2:cr²]:rip13^Irs2, and WT male mice at 8 weeks; average ± SE was determined from 5 animals of each genotype. No tg, no transgene.