Ligand-independent tyrosine phosphorylation in wt and CD19^-/- B-cell precursors. Wild-type and CD19^-/- B-cell precursors were grown in BM cultures. Tonic tyrosine phosphorylation was determined in lysates of total cells or in lysates of sorted IgM^- B cells (5 × 10⁶ cell equivalent/lane). (A) Immunoblot analysis of total tyrosine phosphorylation in cell lysates. Membranes were probed with antiphosphotyrosine, stripped, and reprobed with anti-Igα-specific antibody. Blots shown are representative of 4 different experiments. (B) Lane densitometry analysis of tyrosine phosphorylation in lysates of total cells. Tyrosine phosphorylation lanes shown in panel A were subjected to whole-lane densitometry analysis. Plots represent the obtained signal strength on the y-axis compared with its relative location on the x-axis. (C) Igα phosphorylation was determined by immunoprecipitation (10 × 10⁶ cells), as described in “Materials and methods.” Membranes were probed with antiphosphotyrosine, stripped, and reprobed with anti-Igα-specific rabbit antibodies. The blot shown is a representative of 3 different experiments. (D) Relative phosphorylation of Igα was determined in each experiment by densitometry analysis, where values obtained for a phosphorylated band were divided by the values corresponding to the total band. ▦ indicates CD19^-/-; □, wt. Summarized results of the 3 experiments are shown in a histogram as mean ± SE (right); asterisk indicates values that are statistically different (P < .05). (E) Erk phosphorylation was determined in cell lysates using specific antibodies to phosphorylated Erk and total Erk. Blots shown are representative of 4 different experiments.