The Syk interactome in AML cells. (A) Shown is a schematic representation of the protein purification and identification process. KG-1 cells that were treated with shRNA specific for Syk were cultured in light SILAC medium and served as the negative control. Wild-type KG-1 cells were grown in heavy SILAC medium. Following immunoprecipitation of Syk from both cell batches, precipitated proteins were mixed in equimolar amounts, separated by 1D-PAGE, digested by trypsin, and then analyzed by mass spectrometry. (B) An immunoblot of cleared cellular lysates derived from wild-type KG-1 cells (lane 1) and KG-1 Syk knockdown cells (lane 2) with antibodies against Syk (upper panel) is shown. Protein loading was monitored by anti-actin immunoblotting (lower panel). (C) All identified proteins were plotted according to their signal intensities and their heavy versus light ratio of enrichment (H/L) on logarithmic scales. Red dots indicate proteins with an H/L ratio > 5 that were identified as interaction partners of Syk. A complete list of proteins and statistics is listed in supplemental Table 2. (D) KG-1 cells (left panel) and primary AML cells (middle and right panels) were lysed and subjected to immunoprecipitations (IPs) using anti-Syk antibodies (lanes 2, 4, and 6) or isotype-matched control antibodies (C; lanes 1, 3, and 5). Obtained proteins were analyzed by immunoblotting with antibodies directed against STAT3 and STAT5 (upper and middle panels). Effective IP of Syk was confirmed by immunoblotting using Syk-specific antibodies (lower panels). (E) Recombinant GST or GST-STAT3-SH2 proteins were subjected to lysates of KG-1 cells. Upon affinity purification, eluates were collected and analyzed by immunoblot analysis using antibodies specific for either Syk (upper panel) or GST (lower panel).