NIK expression parallels IKK activity after CHX or MG132 treatment. B5 cells transduced with the control vector (EV1) or B5 cells expressing wild-type NIK (NIK#1) were treated for 4 hours with either vehicle (ethanol, EtOH), cycloheximide (CHX; 50 μg/mL), or MG132 (20 μM). Cytoplasmic extracts were subjected to immunoprecipitation with IKK1-specific antibody, and then immunoprecipitates were used for an in vitro kinase assay. IKK1 expression in the immunoprecipitates was revealed by immunoblotting with IKK1-specific antibody. NIK and actin levels in the cytoplasmic extracts used for immunoprecipitation were determined by immunoblotting with anti-NIK or antiactin antibodies, respectively. IB indicates immunoblotting; IP, immunoprecipitation; GST, glutathione-S-transferase tag.