Fas regulates CD40-induced RAG expression and prevents nuclear localization of CD40. Th40 cells from NOD mice were either isotype-treated (Isotype) or CD40−, Fas− or CD40+ Fas-stimulated (40/Fas; 1C10 and Jo2, respectively) overnight, then nuclear extracts were prepared. (a) RAG1 and RAG2 were analyzed in western blots. (b) CD40 was analyzed in western blots using a C-terminally (sc-975) or an N-terminally (sc-977) reactive antibody. As a control for purity of the nuclear extract, Hsp70, a cytoplasmic protein, was assayed in the two fractions (d0 indicates immediately ex vivo). (c) Cells were treated as above then cytoplasmic and nuclear extracts were subjected to immunoprecipitation of CD40. CD40 levels were analyzed using both a C-terminally (sc-975) and an N-terminally (sc-977) reactive antibody combined. Experiments were done three times. NOD, non-obese diabetic.