BTK signaling networks in AML. (A) Venn diagram showing the number of significantly regulated pTyr sites after 1 hour of ibrutinib treatment. (B) Bar diagram outlining the concordantly regulated pTyr sites in KG-1 and MV4-11 cells. (C) Phosphorylation of Tyr 694 of STAT5 was monitored by using an in vitro kinase assay. The biotinylated peptide encompassing the amino acids TPVLAKAVDGYVKPQIK for STAT5 was used as substrate for BTK obtained from untreated KG-1 cells (upper panel) or for enzymatically active recombinant BTK (lower panel). Tyrosine phosphorylation of these peptides was monitored by anti-phosphotyrosine staining with enzyme-linked immunosorbent assay. *P < .05 between the different treatment groups compared with the controls using Student t test. (D-E) The signaling networks contain proteins that were identified as being phosphorylated (red) or dephosphorylated (green) on tyrosines in response to BTK inhibition. Proteins were grouped by Cytoscape software according to their known protein-protein interaction status listed in the STRING database. The assigned protein functions were derived by manual annotation using Uniprot, PhosphoSitePlus, and PubMed databases. (F) Cleared cellular lysates derived from KG-1 and MV4-11 cells that had either been left untreated or had been treated with 500 nM ibrutinib for 1 hour were subjected to immunoblotting using antibodies against pSTAT5, pERK, pSTAT3, pFOXO1, and phospho-c (pc)-CBL.