Activation mechanisms of Syk in AML cells. (A) Lysates of KG-1 cells that were left untreated (−; lanes 1 and 3) or treated with Bay 61-3606 for 1 hour at a final concentration of 250 nM (+; lanes 2 and 4) were subjected to IP with antibodies against Syk (left panel) or SLP65 (right panel). Subsequently, immunoblot analyses of the obtained proteins were performed by using antiphosphotyrosine antibodies (4G10) (upper panels) and, in addition, antibodies against Syk and SLP65 (lower panels). (B) KG-1 cells were left untreated (−) or were treated with the Src-kinase inhibitor pp2 for 1 hour at a final concentration of 1 µM. The respective lysates were subjected to IP with antibodies against Syk, followed by immunoblot analyses of the proteins obtained; the immunoblot employed antibodies against phosphotyrosine (upper panel) or Syk (lower panel). (C) KG-1 cells were left untreated (lane 2) or were treated with dasatinib (4 nM) and Bay 61-3606 (250 nM) (lane 3), or dasatinib (4 nM) (lane 4) for 1 hour. The respective lysates were subjected to IP with antibodies against Syk, followed by immunoblot analysis of the proteins obtained; the immunoblot employed antibodies against phospho-Syk (pSyk; upper panel) or Syk (lower panel). Isotype-matched antibodies were used as the control (lane 1). (D) Immunoblot analysis was performed with anti-pSTAT3, anti-pSTAT5, and anti-actin antibodies of lysates derived from untreated KG-1 cells (lane 1), KG-1 cells that were treated with either dasatinib (4 nM) and Bay 61-3606 (250 nM) (lane 2), or dasatinib (4 nM) for 1 hour (lane 3). (E) Lysates of KG-1 cells were left untreated (left panel, lane 1; right panel, lanes 3 and 4) or were treated with the phosphatase CIP for 30 minutes (left panel, lane 2) and subsequently subjected to IP with antibodies specific for the Fc-γ chain (left panel, lanes 1 and 2; right panel, lane 4) or nonspecific control antibodies of the same isotype (right panel, lane 3). The proteins obtained were analyzed by antiphosphotyrosine antibodies (left panel, upper immunoblot) or by antibodies against Syk (right panel, upper immunoblot). Effective immunoprecipitation of the Fc-γ chain was confirmed by anti–Fc-γ chain immunoblotting (left and right panels, lower immunoblots). (F) Lysates of KG-1 cells that were left untreated (left panel, lane 1) or were stimulated for 1 or 5 minutes through FcγRI (left panel, lanes 2 and 3) were subjected to IP with antibodies against Syk, followed by immunoblotting using antibodies against pTyr525/526 of Syk (left, upper panel) and Syk (left, lower panel). Shown are immunoblot analyses of lysates derived from KG-1 cells that were left untreated (right panel, lane 1), stimulated via FcγRI for 2 or 5 minutes (right panel, lanes 2 and 3, respectively), or stimulated for 5 minutes after a 1-hour treatment with the Syk inhibitor Bay 61-3606 (right panel, lane 4). Immunoblotting was performed by using phosphosite-specific antibodies against pTyr 694 of STAT5 and pTyr 705 of STAT3 (right, upper and middle panels). Protein loading was monitored by the immunoblotting of actin (right, lower panel). (G) Lysates of KG-1 cells that were left untreated (left panel, lane 1) or were stimulated for 1 or 5 minutes through the integrin receptor Mac-1 (left panel, lanes 2 and 3, respectively) were treated as described in (D). Shown are immunoblot analyses of lysates derived from KG-1 cells that were left untreated (right panel, lane 1), stimulated by the integrin receptor Mac-1 for 2 or 5 minutes (right panel, lanes 2 and 3, respectively), or stimulated for 5 minutes after a 1-hour treatment with the Syk inhibitor Bay 61-3606 (right panel, lane 4). Immunoblotting was performed as described in (D).