P-sites in myeloid Syk. (A) Cleared cellular lysates of Syk-deficient DT40 cells (lane 1), KG-1 cells (lane 2), primary AML cells (lanes 3 and 4), and wild-type DT40 cells (lane 5) were subjected to western blot analyses with antibodies against Syk (upper panel). Protein loading was monitored by anti-actin immunoblotting (lower panel). (B) Shown is the domain structure of human Syk, including the p-sites that were identified by mass spectrometry upon TiO₂-based phosphopeptide enrichment. Phosphorylated tyrosine residues are marked in red, phosphorylated serine/threonine residues in blue. (C) The amino acid sequence of human Syk is shown with p-sites labeled as follows: All p-sites, which were identified in human AML cells, are labeled with red, bold, lowercase letters. Red, bold, capital letters indicate activation-inducing tyrosines that were identified. Bold, italic letters are used if a p-site could not be allocated to 1 of 2 adjacent amino acids. (D) Cell type–specific phosphorylation patterns are outlined. The red circle represents p-sites that were identified in AML cells in this study, while the black circle represents the total number of p-sites that were identified in B cells in previous studies.²⁶  (E) Shown are growth curves of KG-1, FFM05, and FFM12 cells that were left untreated (upper panel, black lines) or were treated with the Syk inhibitor Bay 61-3606 at a final concentration of 250 nM (upper panel, gray lines). Shown are growth curves of KG-1, FFM05, and FFM12 cells that were treated with either control shRNA (lower panel, black lines) or Syk-specific shRNA (lower panel, gray lines). Proliferation was monitored by XTT-based assay. (F) FFM05, FFM12, and KG-1 cells were treated with control- or Syk-specific shRNA and were analyzed for CD34 expression by flow cytometry 7 days after transduction. FITC, fluorescein isothiocyanate; nsp, nonspecific; Syk kd, Syk knockdown.