Figure 8.

Insulin signaling in the heart. The hearts were removed from the same 8 month-old fasted (F) and refed (RF) mice as in Figure 4. Western analysis was performed by immunoblotting (Ib) with antibodies against phospho-IRβ (A, α-pIRβ), phospho-Akt (C, α-pAkt), phospho-eNOS (D, α-peNOS), and phospho-MAPKinase (E, α-pMAPK), in addition to immunoblotting (Ib) with antibodies against IRβ, Akt, eNOS, and MAPK, respectively for normalization. In (A), the lower half of the membrane was immunoblotted with α-tubulin to normalize against protein loading (lower gel). In (B), some aliquots were subjected to immunoprecipitation (Ip) with α-IRS1 antibody followed by immunoblotting wih α-phospho-IRS1 antibody (α-pIRS1, top gel), normalized to total IRS1 (lower gel). The immunopellet was also immunoblotted with α-pIRβ antibody to detect binding between IRS1 and IRβ (middle gel). In (F), a similar co-immunoprecipitation experiment was carried out to detect phospho-CEACAM1 (pCC1) (top gel) or IRβ (middle gel) in the Shc immunopellet. The apparent molecular weight (kDa) is indicated at the right hand-side of each gel. Gels represent more than two separate experiments performed on different mice/genotype/treatment group.