Figure 1

Establishment of NIH/3T3 cells with stable overexpression of functional IRS-1. (A) Wild-type NIH/3T3 cells were serum starved overnight and then treated with serum free DMEM or with 100 nM insulin in serum free DMEM for 20 min. Endogenous IRS-1 was examined by Western blotting. (B) Cells selected after infection by the control retrovirus and those encoding for IRS-1were serum starved overnight and then treated with serum free DMEM or with 100 nM insulin in serum free DMEM for 20 min. The amounts of endogenous and overexpressed IRS-1, and p70 S6K signaling were determined by Western blotting. We used serum free DMEM to avoid the confounding effects on p70 S6K signaling by other growth factors that may be present in serum. (C) Cells were serum starved overnight and then treated with serum free DMEM or with 100 nM insulin in serum free DMEM for 20 min. Akt signaling was monitored by Western blotting. Using serum free DMEM avoided the confounding effects of other growth factors that may be present in serum. (D) Cells were cultured for 18 h before beginning an experiment. The medium was not changed for the control cell group, but was replaced with fresh complete culture medium for the experimental cell group. The ERK1/2 signaling pathway was analyzed using Western blot analysis.