Fig. 4. CD40 ligation induces nuclear translocation of p52 dimers. (A) Nuclear and cytoplasmic fractions were prepared from CD40-293 cells stimulated as indicated and western blotted. (B) Nuclear fractions were prepared from CD40-293 cells stimulated as shown. NF-κB DNA-binding activity was analysed by EMSA. (C) EMSAs were carried out on nuclear extracts from CD40-293 cells stimulated for 0.5 or 6 h with anti-CD40 mAb. Extracts were supershifted with the indicated antibodies to different Rel proteins or pre-immune serum (PI). The position of a p52-containing supershifted NF-κB complex is shown (asterisk). Specificity of κB binding was determined by competition with 100-fold unlabelled κB oligonucleotide (Oligo, NF-κB) or control Oct-1 oligonucleotide (Oligo, Oct-1). (D) Nuclear extracts from CD40-293 cells were supershifted with anti-p100N antibody and analysed by EMSA. The position of supershifted complexes is shown (arrow).