Intact plasma membrane localization and a functional kinase domain of Btk are essential for Btk function after TREM-1 triggering. (A) Schematic illustration of Btk_insB#1 mutants introduced into U937-TD-B#1 cells. (B) Whole cell lysates of U937-TD–derived cells were analyzed by immunoblotting with anti-Btk and anti–β-actin or anti–α-tubulin mAbs. LumiImager signals of the ratio between Btk and the indicated housekeeping protein are displayed as mean ± SD from 7 independent experiments. (C, D) U937-TD–derived cells were incubated with either plate-bound anti–FLAG or isotype-matched control mAbs or PMA. (C) Supernatants were collected after 8 (TNF-α) or 16 (IL-8) hours and cytokine production was analyzed by ELISA. Data are displayed as mean ± SD of at least duplicates of ELISA from duplicate cultures. (D) Expression analysis of CD11c and CD86 after 48 hours by flow cytometry is depicted. Data are displayed as mean ± SD from duplicate cultures. (E) U937-TD derived cells were stimulated with the anti-FLAG mAb for 5 minutes. Postnuclear supernatants were analyzed by immunoblotting with anti–pErk1/2 and anti-Erk1 mAbs. LumiImager signals were quantified, and the ratio between pErk1/2 and Erk1 was calculated. (F) Ca²⁺ mobilization of Indo-1 AM-labeled U937-TD–derived cells was analyzed by flow cytometry. Cells were stimulated with the anti-FLAG mAb at the time point indicated by the arrow. One representative experiment of 7 (B), of 4 (C,D), of 3 (E) and of 5 (F) independent experiments is shown. *P < .05 and **P < .003 (Student t test).