Methods

Cell Culture. 3T3/HIR fibroblasts (Whittaker et al., 1987) were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. In Vivo3 Phosphorylation and Immunoprecipitation of In-sulin Receptors. Cells were incubated for 1 h in phosphate-free Dulbecco’s modified Eagle’s medium at 37 C, at which time [32P] orthophosphate was added to a final concentration of 2 mCi/mL. Incubation was continued for an additional 2 h. Cells were then incubatedwith or without 100 nM PMA for 20 min at 37 C, and the cultures were placed on ice and solubilized in 10 mM Hepes, pH 7.8, containing 1% Triton X-100, 500 mM NaCl, 5 mM EGTA, 30 mM sodium pyro-phosphate, 50 mM sodium fluoride, 100 mM sodium ortho-vanadate, 0.1% bovine serum albumin, 10 Mg/mL leupeptin, and 0.1 mM phenylmethanesulfonyl fluoride (bufferA). In-soluble matter was removed by centrifugation for 15 min at 15 000 rpm in a microfuge. Cell supernatants were added to aIR-1, a monoclonal antibody against the insulin receptor (Kull et al., 1983), which was prebound to protein A-Se-