Expression of Btk insensitive to the B#1 shRNA restores the defects of the Btk knockdown cell line. (A) A schematic picture of the construct used to prepare the Btk rescue cell line, U937-TD-B#1-Btk_insB#1WT, is depicted. (B) Whole cell lysates of U937-TD–derived cells were analyzed by immunoblotting with anti-Btk and anti–β-actin mAbs. LumiImager signals of the ratio between Btk and β-actin 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) ELISA analysis of supernatants collected after 8 (TNF-α) or 16 (IL-8) hours. Data are displayed as mean ± SD from ELISA performed at least in duplicates of duplicate cultures. (D) Expression analysis of CD11c and CD86 after 48 hours by flow cytometry is shown. 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 Abs. 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 5 (C,F), of 4 (D) and of 3 (E) independent experiments is shown. *P < .05 and **P < .005 (Student t test). ins indicates insensitive; and WT, wild-type.