Figure 2.

NFκB EMSA. EMSA of nuclear extracts from wt and Cblb^−/− CD4⁺ T cells, unstimulated or stimulated overnight with 3 μg/ml platebound anti-CD3 and 1 μg/ml anti-CD28. As wt oligo, the NFκB consensus sequence within the minimal GM-CSF promoter (A) or predicted NFκB sites in the distal enhancer element of the IL-3/GM-CSF gene cluster “CNSa” (B,C) were used. To validate binding specificity, mutated oligos were used instead. Wt and mutated sequences were added in excess as unlabeled competition oligos (cold comp., cold mut.). Where indicated, an anti-p50 antibody was added. One representative experiment out of three (A) or two (B,C) is shown.