EμB29-Btk gene therapy restores Btk-dependent BCR and mitogen signaling. BM and splenocytes cells were assessed using various functional assays at the time of harvest. (A) In 1 of 4 experiments, splenocytes were stained for surface markers, stimulated with anti-IgM or ionomycin, and analyzed for calcium flux. Representative traces from B220⁺ cells in WT mock (n = 1), KO mock (n = 1), and MOI 15 to 25 (n = 4) experimental groups are shown. (B) Splenocytes were analyzed for cell proliferation in response to anti-IgM, LPS, or PMA/ionomycin stimulation. (C) BM or splenocytes cells were analyzed for colony-forming unit cell production. WT and KO datasets include both unmanipulated and mock-transplanted animals. Data represent 4 independent experiments, unless otherwise noted. P values compare EμB29-Btk vs KO groups: ***P < .001; **P = .001-.01; *P = .01-.05.