Figure 1.
NF-κB/Rel DNA binding and translocation is decreased in anti-IgM–activated, btk-mutated, xid B cells. (A) Splenic B cells from wild-type and xid mice were cultured for 4 h in medium, F(ab′)2 anti-IgM (10 μg/ml), or CD40 ligand (1:2 dilution), cells were harvested, nuclear extracts prepared, and EMSAs performed as described in Materials and Methods. This figure represents one of three experiments. Densitometry was performed on the upper band of the gel shift. Addition of a cold competitor (100-fold excess) demonstrated the specificity of NF-κB binding (data not shown), as shown previously 49. (B) Western analysis of nuclear c-rel expression (top panel) was conducted using 20 μg of extract prepared from wild-type or xid splenic B cells treated as described in A. Transcription factor SP-1 served as a nuclear loading control (bottom panel). Densitometry was performed. After normalization to the loading control, it was determined that anti-IgM treatment induced nuclear c-rel 4.8-fold in wild-type and 0.75-fold in xid B cells. This figure represents one of five experiments.