Btk becomes phosphorylated after TREM-1/DAP12 triggering. (A) U937-TD cells were stained with anti–TREM-1, anti-FLAG or anti–HLA-I (open histograms) or isotype-matched control mAbs (filled histograms) followed by staining with goat anti–mouse-PE Abs and analyzed by flow cytometry. (B) U937-TD cells were incubated with the indicated stimuli for 5 minutes at 37°C. pY proteins were immunoprecipitated from postnuclear supernatants using covalently coupled IgG2b-Sepharose followed by anti–pY-Sepharose. Phosphorylation was detected by immunoblotting with anti-Btk pY551 and anti-Btk Abs. (C) Human PBMCs containing 14% TREM-1⁺ monocytes were incubated with the indicated stimuli for 2 minutes. Btk was immunoprecipitated from postnuclear supernatants using the anti–Btk Ab and Protein A Ultralink Resin. Immunoblotting was performed with anti-Btk pY551 and anti–Btk Abs. (D) U937-TD cells transduced with Myc-tagged Btk were placed in medium alone or in the presence of DMSO or the Src kinase inhibitor, PP2, or the Syk inhibitor IV for 20 minutes at 37°C. Subsequently, cells were incubated with the indicated stimuli for 5 minutes at 37°C. Btk was immunoprecipitated from postnuclear supernatants using the anti–Myc-tag mAb and Protein A Ultralink Resin. Immunoblotting was performed with anti-Btk pY551 and anti-Btk Abs. IP indicates immunoprecipitation; and pY, phosphotyrosine.