Rapid activation of the NF-κB and JNK pathways by Bcl10 but not by its CARD. (A-D) NF-κB activation. Bcl10-ER or Bcl10 CARD-ER-expressing cells or a nonexpressor control clone were treated with E2 (1 μM) for indicated periods of time (minute) and directly lysed with sodium dodecyl sulfate (SDS) sample buffer. Lysates were blotted with anti-IκB-α (A) and, after stripping, with anti-IκB-β (B) to detect degradation of IκB-α or IκB-β. To control for sample loading, identical gels were blotted with anti-β-tubulin (C). (D) Indicated cells were treated with E2 (1 μM) to activate Bcl10 or Bcl10 CARD. Nuclear lysates were prepared and electrophoretic mobility shift assay (EMSA) performed with a ³²P-labeled oligonucleotide consensus sequence for NF-κB binding. (Right) The specificity of the assay was verified in a repeat experiment in which competing nonlabeled NF-κB- or activator protein 1 (AP1)-binding oligonucleotides (the latter being a negative control) were included in 25-fold molar excess during the binding. (E-G) JNK activation by Bcl10 but not its CARD domain. Indicated cells were treated with E2 (1 μM) and analyzed for JNK activation by blotting the lysates with an antibody specific for phosphorylated JNKs (arrows) (E). CD40-stimulated (15 minutes) WEHI-231 cells were used as a positive control. To verify equal loading of samples, an identical gel was blotted with anti-β-tubulin (F). (G) Cells were treated with E2 (1 μM) and lysed. JNK1 was immunoprecipitated from the lysates and measured for its in vitro kinase activity using purified glutathione S transferase (GST)-c-Jun (N-terminus) as the substrate. CD40-stimulated (15 minutes) nonexpressor cells were used as a positive control for the kinase assay. The kinase reactions, once no longer radioactive, were blotted with anti-JNK1 to confirm comparable levels of immunoprecipitated JNK1 protein in the reactions (bottom).