Figure 4.
Increased nuclear levels and activity of c-Rel and elevated Bcl-xL expression in activated DR6−/− B cells. (A) Western blot analysis of c-Rel in cytoplasmic and nuclear extracts from DR6-deficient (−/−) and WT (wt) B cell cultures. Purified splenic B cells were stimulated with or without 10 μg/ml anti-IgM or 1 μg/ml anti-CD40 for 30 min or 4 h as indicated. Cytoplasmic (C) and nuclear (N) extracts were prepared as described in Materials and Methods. C-Rel was detected with specific anti–c-Rel Ab after SDS-PAGE. Equal amounts of either cytoplasmic or nuclear protein extracts were loaded into wells and data shown are representative of two independent experiments. (B) GMSA with nuclear fractions of DR6−/− and WT B cells activated with anti-IgM or anti-CD40 for 0.5 or 4 h as described in Materials and Methods. Nuclear c-Rel from activated DR6−/− B cells bound to its specific DNA probe (top) and supershifted with the addition of anti–c-Rel antibody (bottom). Middle bands were nonspecific binding, as they were not competed by cold homologous oligonucleotide. (C) B cells from DR6-deficient (−/−) or WT (wt) were either untreated or treated with anti-IgM or anti-CD40 for 4 h and total cell lysates were prepared in 1× RIPA buffer as described in Materials and Methods. After SDS-PAGE and Western transfer, Bcl-xL in anti-CD40–stimulated samples was detected with rabbit polyclonal anti–Bcl-xL, whereas mouse monoclonal anti–Bcl-xL was used to detect the protein in anti-IgM–treated samples as the stimulus was a rabbit anti–mouse IgM antibody that was detected by the original secondary, anti–rabbit antibody. All blots were probed with mouse anti–β actin as a loading control. Data shown are representative of two independent experiments.