FIG. 5.

c-Rel associates with IκBα in mature B cells but not in pre-B cells and mature T cells. (A) Whole-cell extracts (100 μg) from mature B cells (A20), pre-B cells (70Z), or mature T cells (D5h3) were immunoprecipitated with nonspecific IgG (lane 1), anti-IκBα antibody (lane 2), or anti-IκBβ antibody (lane 4). For sequential immunoprecipitation, extracts were precleared with the first antibody and immunoprecipitated with the second antibody, as indicated above the lanes (lanes 3 and 5). Immunoprecipitates were separated by SDS-PAGE, and Rel proteins associated with IκBs were detected by immunoblotting. Left panel shows the results using anti-c-Rel and anti-IκBα antibodies (Ab) to probe the blots; right panel was probed with anti-p65 and anti-IκBα antibodies. IκBβ protein was not assayed for because it comigrated with the Ig heavy-chain protein used for immunoprecipitation. Results are representative of at least three independent experiments. (B) Whole-cell extracts from indicated cells were analyzed by immunoprecipitation using nonspecific IgG (lane 1), anti-IκBα (lane 2), and anti-IκBβ (lane 3), followed by immunoblotting as described above. For each cell type, the upper panel shows the results using anti-c-Rel antibodies to probe the blots, and the lower panel shows the results of probing with anti-p65 antibodies. Top three rows correspond to cell lines representing pre-B, mature B, and T cells. Bottom row shows the analysis of primary splenic B and T cells purified as described in the text. Results are representative of at least two independent experiments with each cell type.