Expression analysis of CD40 mutants. (A) Surface expression of CD40 in BLCLs from control (C) and from P1, P2, P4, and P5 patients, analyzed by flow cytometry with mAb 5C3-PE conjugated (top panels) and mAb 626.1 (bottom panels). Negative staining is shown as shaded peaks. (B) Total expression of CD40 analyzed by immunoblot with a polyclonal anti-CD40 antibody (H120), comparing CD40 expression in controls (C; primary B cells; Daudi cell line), heterozygous carriers (P2′m, P2′ mother; P5′m, P5′ mother) and mutants (P1; P2; P4; P5). T cells were used as negative control. Immunoblot for β-actin is shown as loading control. (C) CD40 mRNA expression in controls (C and C'; primary B cells; Daudi and Jurkat cell lines) and in patients' BLCLs, analyzed by real-time PCR using 2 Taqman assays designed on exons 1-2 (dark gray) and exons 5-6 (light gray) junctions. Expression values, relative to β-actin, are normalized to the expression observed in (C). Each analysis was performed with triplicate samples, and data are shown as means ± SD.