TREM-1 signaling is impaired in Btk knockdown cells. (A) Whole cell lysates of U937-TD–derived cells were analyzed by immunoblotting with anti-Btk and anti–β-actin mAbs. LumiImager signals of Btk and β-actin were quantified and the ratio between Btk and β-actin was calculated. (B-C) U937-TD–derived cells were incubated for 16 (B) or 48 (C) hours with either plate-bound anti-FLAG or isotype-matched control mAbs or PMA. (B) Supernatants were analyzed by ELISA for TNF-α and IL-8. Data are displayed as mean ± SD of at least duplicates of ELISA. (C) Cells were analyzed for expression of the differentiation/activation markers, CD11c and CD86, by flow cytometry. (D) U937-TD–derived cells were incubated with medium alone or with isotype-matched anti-HLA-I or with anti-FLAG mAbs for the indicated time periods. Postnuclear supernatants were analyzed by immunoblotting with anti-pErk1/2 and anti-Erk1 Abs. LumiImager signals were quantified, and the ratio between pErk1/2 and Erk1 was calculated. (E) U937-TD cells were incubated with medium, isotype-matched anti–HLA-I or with anti-FLAG mAbs in the presence or absence (w/o) of LY294002 or DMSO for 5 minutes. Postnuclear supernatants were analyzed by immunoblotting with anti–pErk1/2 and anti–β-actin Abs. Signals were quantified, and the ratio between pErk1/2 and β-actin was calculated. (F) Ca²⁺ mobilization of the Indo-1 AM-labeled U937-TD–derived cells was analyzed by flow cytometry. Cells were stimulated with mAb at the time point indicated by the arrow. (G) U937-TD–derived cells were stimulated with isotype-matched anti–HLA-I or anti-FLAG mAbs for the indicated time periods. Postnuclear supernatants were analyzed by immunoblotting with anti-PLCγ1 pY783 and anti-PLCγ1 Abs. One representative experiment of 2 (G), of 3 (A-F), of 7 (B), of 5 (C) and of 4 (D) independent experiments is shown. n.d. indicates not detectable.