The V_H81X Tg prevents tumor formation in Btk/Slp65 double-deficient mice. (A) Quantitative RT-PCR analyses of c-Myc and N-Myc expression in Slp65^−/− (n = 16) and Btk^−/−Slp65^−/− (n = 27) pre-B cell tumors normalized with GAPDH. Values in wild-type pre-B cells, cultured for 5 days with 100 U/mL IL-7, were set to 1. For each of these groups, the horizontal line represents the mean of the relative expression level. (B) Effect of the V_H81X Tg on pre-B cell proliferation in vivo. The absolute numbers of BrdU⁺ CD19⁺cμ⁺cκ⁻ pre-B cells in the BM, 4 hours after intraperitoneal injection of a single dose of BrdU, as determined by flow cytometry. Average values and SEM of 3 animals per group are shown. (C) Effect of the V_H81X Tg on pre-B cell proliferation in vitro. Results are displayed as fold expansion after 5 days of culture in the presence of IL-7 (100 U/mL), whereby the pre-B cell numbers at the start of the culture where set to 1. Bars represent average values and SEM of 7 to 8 animals per genotype. (D) Syk phosphorylation in pre-B cell cultures from non-V_H81X Tg Slp65^−/− and V_H81X Tg Btk^−/−Slp65^−/− mice. Cells were either unstimulated (−) or stimulated for 5 minutes with polyclonal anti-IgM F(ab)2 fragments. The presence of phosphorylated Syk was detected in antiphosphotyrosine immunoprecipitates from total cell lysates, analyzed by Western blotting by the use of Syk-specific antibodies. (E) Kaplan-Meier tumor-free survival estimates for Btk^−/−Slp65^−/− mice (n = 20) and V_H81X transgenic Btk^−/−Slp65^−/− mice (n = 25).