Fig. 2: Differential phosphoproteomic signatures of DKO, IR, K1030R, ∆CT, and JMO cells.

a T-distributed stochastic neighbor embedding (t-SNE) analysis of the phosphosites identified by LC–MS/MS from DKO, IR, K1030R, ∆CT, and JMO cells in the basal and insulin-stimulated states. Cells were serum starved for 6 h with DMEM containing 0.1% BSA before 100 nM insulin stimulation for 15 min. b Quantification of exemplary phosphorylation events in IRS1 and Shc1. Data are means ± SEM of phosphosites intensity values (×10⁴) (n = 2–3). P-values are basal vs. insulin, two-way ANOVA followed by Šídák’s multiple comparisons test. c Heatmap showing the hierarchical clustering of the phosphopeptides in DKO, IR, K1030R, ∆CT, and JMO cells in the basal and insulin-stimulated states. Values are Z-scores of log2 transformed intensity values. d REACTOME pathway enrichment analysis of phosphosites in the Up by Insulin cluster. The functional enrichment analysis was tested by the STRING database, where FDRs were calculated using the Benjamini–Hochberg procedure. Plots are −log10 transforms of enrichment FDR value. e Quantitation of exemplary phosphosites in the Up by Insulin cluster. Data are means ± SEM of phosphosites intensity values (×10⁴) (n = 2–3). P-values are basal vs insulin, two-way ANOVA.