Figure 4.

Btk functional deficiency alter MTOC polarization, B cell activation and proliferation. (A) Immunoblot of WT, Xid, and Ibru-B cells stimulated with Ab-coated plates, probed with the specified antibodies. Quantification of phosphorylated ERK1/2 (p-ERK1/2, Y202/Y204), phosphorylated PCKα/β (p-PKCα/β, T638/641) and phosphorylated Akt (p-Akt, S473) intensity normalized to tubulin (Tub; loading control) by densitometry at each time point; right. (B) DIC and fluorescence γ-tubulin (γ-tub; green) images are shown of representative B cell/pseudo-APC conjugates, fixed at 30 min, for each indicated condition. Dashed circle, pseudo-APC; Bar, 2 μm. (C) Frequency of B cells as in B, in the specified polarity index (PI) groups. The PI per B cell was estimated as the ratio of A and B distances (explicative diagram, left); data shown as mean ± SD of 90 B cells in each case. (D–E) B cells were co-cultured with pseudo-APC, unloaded (none) or su-Ag-loaded (20 molec/μm²; su-Ag-20) at 1:1 ratio, 20 h. (D) Representative profiles of CD69, CD25 and CD86 for each case. (E) Frequencies of B cells expressing CD69, CD25 or CD86 (left) and mean fluorescence intensity (MFI) values for these markers (right) in each condition and for each B cell type. (F–G) Violet-labeled B cells were co-cultured with pseudo-APC, unloaded (none) or su-Ag-loaded (20 molec/μm², su-Ag-20, ratio 1:5; 100 molec/μm², su-Ag-100, ratio 1:1), with IL-4, 96 h. (F) Representative profiles of violet-tracer in each case. (G) Frequencies of cycling B cells (with diluted violet-tracer level) in each condition and for each B cell type. Data in (A) are the mean ± SD of three experiments (n = 3); data in (C) are the mean ± SD of two experiments (n = 2), and in (E,G) are the mean ± SD of three experiments (n = 3). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001 by Student's t-test with WT in each case.