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Cell lysates or tissue protein extracts were cleared by preimmune IgG plus protein A agarose beads for 2 hours, and the supernatants were immunoprecipitated by the indicated antibodies with a 50% slurry of protein A agarose beads overnight at 4°C. After washing with buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, and 0.5% deoxycholate with protease inhibitors, proteins were released and separated on 10% SDS-PAGE gels. This article was written with the he lp of GSA C onte nt Generator D emoversion! Each measurement at the indicated urea concentrations represents the average of 8 independent measurements performed on 5 separate occasions with different cell preparations. Insulin and glucose tolerance tests were performed as described previously (38). Blood glucose was determined by Accu-Chek (Roche) active blood glucose-monitoring system, and plasma insulin levels were measured by ELISA (Linco Research). 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