IGFBP7 prolongs IRS, AKT, and ERK activation by insulin in ALL cell lines. (A) ELISA results for Phospho-IRS-1 (pan-Tyrosine; #7133, Cell Signaling Technology) on different ALL cell lines that were starved for 4 hours in serum free RPMI-1640 medium and then left untreated (Control) or stimulated for 15 minutes or 4 hours with insulin (500 ng/mL) and/or IGFBP7 (100 ng/mL). Bars represent means ± SE for triplicate wells. (B) Western blot results for Phospho-Akt (Ser473) and Phospho-Erk1/2 on the RS4;11 and TALL-1 ALL cell lines stably expressing a scramble shRNA (Scr) or shRNA against IGFBP7 (sh.959), after 4-hour starvation in serum-free RPIM-1640 and left untreated or stimulated for the indicated time with insulin (500 ng/mL) and/or IGFBP7 (100 ng/mL). The effect of IGFBP7 in prolonging (4 hours) AKT and ERK activation is better visualized in sh.959 cells. Of note, IGFBP7 is not needed for the short-term (15 minutes) stimulation of ALL cells with insulin (supplemental Figure 14). Statistical analysis was done by 2-way ANOVA and Bonferroni posttests (*P .05, **P .01, ***P .001, and ****P .0001).