FIG. 6.

Reconstitution of IRS-1 expression in IRS-1-deficient brown adipocytes. (A) IRS-1-deficient brown adipocytes (clone 4) were cultured in the presence of 10% FCS until 60 to 70% confluence was reached. Then cells were transfected with pCMVhis IRS-1^wt and pCMVhis IRS-1^F895 cDNA constructs according to the calcium phosphate-mediated protocol. Twenty-four hours after transfection, histidinol (10 mM) was added to select stable transfectants. Several histidinol-resistant cell lines were obtained, and the expression of IRS-1^wt and the mutant IRS-1^F895 was assessed by Western blot analysis with anti-IRS-1 antibody. (B) Quiescent cells were stimulated with 10 nM insulin for 5 min, and total cell lysates were immunoprecipitated (IP) with anti-IRS-1 or anti-Grb-2 antibodies. The resulting immune complexes were analyzed by Western blotting (WB) with anti-Tyr(P), anti-IRS-1, and anti-Grb-2 antibodies as indicated for each panel. The positions of IRS-1 and Grb-2 are indicated. A representative experiment is shown. The autoradiograms corresponding to three independent experiments, which used two clones of reconstituted cells, were quantitated by scanning densitometry. Results are expressed as arbitrary units of tyrosine phosphorylated IRS-1 and are means ± standard errors.