Figure 8.

Expression of wild-type and mutant human insulin receptor cDNAs in CHO cells. (A) Immunoblot analysis with antibodies to the insulin receptor of partially purified insulin receptors from nontransfected cells (lane 1), from cells transfected with cDNA for the wild-type human insulin receptor (Gly⁹⁹⁶) (lane 2), and from two cell lines transfected with cDNA for the Val⁹⁹⁶ mutant form of the receptor (lanes 3 and 4). (B) Autophosphorylation of partially purified insulin receptors from nontransfected cells (lanes 1 and 2) or cells transfected with wild-type (lanes 3 and 4) or mutant (lanes 5 to 8) human insulin receptor cDNAs as in (A). CHO cells were solubilized and insulin receptors were partially purified on wheat germ agglutinin-agarose. Receptors were incubated in the absence (odd lanes) or presence (even lanes) of 0.1 µM insulin for 30 min and then were phosphorylated in the presence of [γ-³²P]ATP and MnCl₂ as previously descrived.¹⁸⁾ Insulin receptors were then immunoprecipitated and analyzed by SDS-PAGE and autoradiography. Modified with permission from ref. 38.