Differential phosphorylated patterns by IR and IGF1R in the ligand action. (A) REACTOME pathway enrichment analysis of phosphosites in category IIA, i.e., those up-regulated by ligand for IR > IGF1R. Plots are –log10 transforms of enrichment FDR value. (B) Representation of some important phosphosites in selected pathways and quantification of exemplary phosphosites in the cluster of category IIA. Numbers in the center of each circle indicate number of proteins regulated within each pathway. Data are means ± SEM of phosphosite intensity values. ##P < 0.01 basal vs. ligand, **P < 0.01, ***P < 0.001 IR vs. IGF1R, two-way ANOVA. (C) Kinase enrichment analysis for phosphosites in category IIA. Plots are –log10 transforms of enrichment P value. (D and E) Validation of phosphoproteomics by immunoblotting in lysates from IR, IR_IGF1R, IGF1R, and IGF1R_IR cells. Representative immunoblot analysis (D) and quantification of phosphorylation of p-Foxo3aT32, p-mTORS2481, and p-PRAS40T246 (E). Data are means ± SEM (n = 3 per group). #P < 0.05, ##P < 0.01, ###P < 0.001 basal vs. ligand, **P < 0.01, two-way ANOVA. (F) REACTOME pathways enrichment analysis of phosphosites up-regulated by ligand for IGF1R > IR (category IIB). Plots are –log10 transforms of enrichment FDR value. (G) Representation of some important phosphosites on selected pathways and quantification of exemplary phosphosites in the cluster of category IIB. Data are means ± SEM of phosphosites intensity values. ##P < 0.01, ###P < 0.001 basal vs. ligand, *P < 0.05, **P < 0.01, ***P < 0.001 IR vs. IGF1R, two-way ANOVA. (H) Kinase enrichment analysis of phosphosites in category IIB. Plots are –log10 transforms of enrichment P value.