Loss of IGF-1 responsiveness in IGF-1R DN–expressing cells. (A) Western blot analysis of WT and mutant IGF-1R expression in transduced ST4 cells. Total-cell lysates (50 μg) from ST4-E, ST4-WT, and ST4-DN were electrophoresed, electroblotted, and stained with an anti–IGF-1Rα Ab. Membranes were subsequently probed with an antiactin Ab to confirm equal protein loading in each lane of the gel. (B) Flow cytometric analysis of IGF-1Rα surface expression in ST4-E (i), ST4-WT (ii), and ST4-DN (iii) cultured in complete medium with expression of IGF-1Rα shown in the gray histograms and the background of mouse IgG1 negative control in the open histograms. Boxed results show percentage of positive cells and mean specific fluorescence, calculated by subtracting the positivity and the mean of fluorescence obtained with isotype-matched control Ig from that detected with the specific mAb. (C) Western blot evaluation of IGF-1–induced Akt, MAPK, and STAT3 activation on ST4-E, ST4-WT, and ST4-DN deprived of serum for 4 hours treated with IGF-1 (100 ng/mL) for 15 minutes. Phosphorylation was evaluated with anti–phospho-Ser (473)-Akt, anti–phospho-Thr (202)/Tyr (204)–p44/42 MAPK, or anti–phospho-Tyr (705)–STAT3 Abs. Membranes were subsequently probed with anti-Akt, anti-p44/42 MAPK, or anti-STAT3 Abs. All the experiments were performed independently at least 3 times. (D) ST4-E (○), ST4-WT (□), and ST4-DN (▪) were cultured in serum-free medium with scalar doses of IGF-1. After 72 hours, cells were harvested, stained with trypan blue, and counted. The graph illustrates the mean result of 4 independent experiments.