Increased activation of p38 MAP kinase is inversely correlated with LSC activity. (A) MS/MS spectrum (left panel) of phosphopeptide-enriched FLB1 extracts showing phosphorylation on threonine 180 (T180) and tyrosine 182 (Y182) in the activation loop of p38 MAPK. The amino acid sequence is displayed with the corresponding to y-type ion fragments. Ion chromatogram (right panel) of the p38 MAPK phosphopeptide HTDDEMpTGpYVATR (encompassing the activation site) in cytosolic extracts shows an ∼ 3-fold up-regulation in FLB1. Mass spectrometry analyses were performed in biologic triplicates. (B) Western blot analysis of the phosphorylation status of p38 MAPK, Msk1, and Mapkapk2 in FLA2 and FLB1 specimens. (C) Western blot analysis of the phosphorylation status of Erk1/2 and Jnk kinases in FLA2 and FLB1 specimens. (D) Western blot analysis of the phosphorylation status of Akt, mTOR, and S6k1 kinases in FLA2 and FLB1 specimens. (E) Western blot analysis of the phosphorylation status of STAT1/3/5 in FLA2 and FLB1 specimens. α-tubulin was used as a loading control. All Western blots shown are representative results from 3 independent experiments using 5 different samples for each leukemia per experiment.