Immunoblot analysis of Insr expression in different tissues of the transgenic knockouts. (A) Liver. The upper panel represents an autoradiogram showing immunoreactivity with anti-Insr antiserum; the lower panel shows an immunoblot with anti-tubulin antiserum to confirm equal loading of all lanes. We used 3- to 4-month-old animals for these determinations. (B) Islets. For these experiments, we partially purified islets by Ficoll density gradient centrifugation. Please note the prominent band corresponding to the receptor precursor (Insr precursor). To normalize for β-cell content, we used the β cell–specific marker Glut2 (middle panel). We used tubulin to normalize for total protein content (bottom panel). We used 2-month-old animals for these experiments. (C) Widespread transgene expression in brains of L1 Ttr-Insr mice. We obtained specimens from different brain sections and analyzed them by immunoblot. On the left, we present a control obtained with anti–glutamate receptor antiserum (GluR) to normalize for gel loading. On the right, we show immunoblots with anti-Insr antiserum. Arrows indicate the position of the Insr β-subunit. We used 3- to 4-month-old animals for these determinations. (D) Lack of Insr expression in muscle, heart, spleen, and adipose tissue. We show representative blots of 3- to 4-month-old mice. We obtained similar results with specimens from mice of different ages. We could not obtain adipose tissue from L2 and L3 mice because they are lipoatrophic.