Functional characterization of the Syk/STAT interplay. (A) Immunoblot analysis was performed with anti-pSTAT3 and anti-pSTAT5 antibodies (upper panels) or anti-STAT3 and anti-STAT5 antibodies (lower panels) of untreated KG-1 cells (−; lane 1) or KG-1 cells that were treated with the Syk inhibitor Bay 61-3606 at a final concentration of 250 nM for 1 hour (+; lane 2). (B) Immunoblot analysis was performed using the same antibodies as described in (A) of untreated AML cells derived from the primary culture FFM05 (−; lane 1) and AML cells from the same culture, but they were treated with the Syk inhibitor as described in (A) (+; lane 2). (C) Cleared cellular lysates of KG-1 cells treated with unspecific shRNA (lane 1) or Syk-specific shRNA (lane 2) were analyzed by immunoblotting with phosphosite-specific antibodies against the activatory tyrosines of STAT3 and STAT5 (two upper panels). Syk expression and protein loading were monitored by immunoblotting with antibodies against Syk or actin (two lower panels). (D) Three days after shRNA transduction, KG-1 cells were tested for their expression levels of Pim1 and Socs3 by immunoblotting using specific antibodies (upper and two middle panels). Protein loading was monitored by anti-actin immunoblotting (bottom panel). (E-F) Phosphorylation of Tyr 705 of STAT3 and Tyr 694 of STAT5 was monitored using an in vitro kinase assay. For this purpose, the biotinylated peptides encompassing the amino acids EHPEADPGSAAPYLKTKFIC for STAT3 and TPVLAKAVDGYVKPQIK for STAT5 were used as substrates for the Syk obtained from untreated KG-1 cells (left panels) or for the enzymatically active recombinant kinase domain of Syk (right panels). Tyrosine phosphorylation of these peptides was monitored by antiphosphotyrosine staining with enzyme-linked immunosorbent assay.