Constitutive and CD40-stimulated NF-κB activation in BJAB cells expressing MALT1 or API2-MALT1. BJAB cells transfected with pMSCV-IRES-EGFP vector alone or single clones transfected with vector carrying MALT1 or API2-MALT1 were untreated (□) or treated with 0.1 μg/mL cross-linked CD40L for 4 hours (▦). Two micrograms of nuclear protein extracts from each cell line was used in an ELISA assay measuring (A) p50, (B) c-Rel, and (C) p65/RelA subunits of NF-κB. The results represent triplicates of 1 of 3 representative experiments. (D) Enhanced NF-κB activation by API2-MALT1 and MALT1 upon CD40L but not PMA/ionomycin (PMA + IONO) or TNFα stimulation. BJAB cells expressing IκBα reporter only (WT; □) or reporter with MALT1 (▦) or API2-MALT1 (▧) were untreated or treated for 4 hours with TNFα, PMA/ionomycin, or cross-linked CD40L. Lysates were used in a luciferase assay to measure the amount of exogenous IκBα-luciferase, inversely related to the activity of IKK. Each bar represents the reporter level as a percentage normalized to the corresponding untreated cells. Values are obtained from triplicates of one of several representative experiments. Error bars indicate the standard deviations obtained from triplicate samples. The experiment is representative of several independent experiments.