FIG. 9.

Reconstitution of insulin-induced mitogenesis by transient transfection with GFP–IRS-1^wt, but not with GFP–IRS-1^F895, construct. (A) IRS-1-deficient brown adipocytes were transfected with the GFP–IRS-1^wt and GFP–IRS-1^F895 cDNA constructs as described in Materials and Methods. A representative image of fluorescent cells is shown. (B) Quiescent cells were stimulated with 10 nM insulin for 5 min, and total cell lysates were immunoprecipitated (IP) with anti-IRS-1 antibody. The resulting immune complexes were analyzed by Western blotting (WB) with the anti-Tyr(P) antibody. The position of IRS-1 is indicated. A representative experiment is shown. (C) Quiescent cells (IRS-1^+/+ and pGFPIRS-1^wt and pGFPIRS-1^F895 transfectants) were stimulated with insulin (10 and 100 nM) for 5 min, and total cell lysates were submitted to Western blot analysis with the anti-phospho MAPK and anti-MAPK antibodies. A representative autoradiogram is shown. (D) Cells were cultured for 24 h in a serum-free medium either in the absence or presence of insulin (10 to 100 nM). DNA synthesis was determined by [³H]thymidine incorporation (0.2 μCi/ml) over the last 4 h of culture. After two washes with ice-cold PBS, cells were lysed and trichloroacetate-precipitable DNA was then counted for incorporated radioactivity. Results are expressed as disintegrations per minute (dpm) per dish and are means ± standard errors from six independent experiments, each one performed in triplicate. +/+, IRS-1^+/+.