Co-expression of LMP1/2A decreases B cell proliferation and TRAF2 levels compared to LMP1.

<p>(A–C) Purified splenic CD43⁻ B cells were stimulated with combinations of IL-4, LPS, anti-IgM and anti-CD40. Proliferation as measured by ³[H]-thymidine incorporation is shown for 8 experiments with 8 mice per genotype. (D) Levels of TRAF2 and TRAF3 message were compared by RT-PCR using the ΔΔC_T method with HPRT as a housekeeping gene. Fold change in TRAF2 and TRAF3 expression in transgenic B cells compared to wildtype is shown for at least 4 experiments with 9 mice per genotype. (E) Immunoblot analysis shows TRAF2 (∼53 kDa), TRAF3 (∼62 kDa) and GAPDH (∼38 kDa) expression in purified B cells from WT, LMP1, LMP2A and LMP1/2A mice and TRAF2-expressing CHO-K1 cells. Each lane represents a single mouse, and the data are representative of several immunoblot analyses with between 6 and 12 mice per genotype. (F) Quantification of TRAF2 signal normalized to GAPDH and wildtype TRAF2 signal is shown as an average of all mice from each genotype. For a description of quantification, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002662#s4" target="_blank">Methods</a>. LMP1 bars have been placed first as LMP1 B cells gave the largest responses. Data are represented as mean ± standard error. *, <i>P</i><0.05; <i>**, P</i><0.01; ***, <i>P</i><0.001 by one way ANOVA.