NF-κB binding activity and NF-IL6 phosphorylation in TEC after adhesion to normal thymocytes. (A) NF-κB binding activity of cytoplasmic (Cyt) and nuclear (Nuc) extracts of TEC untreated, cocultured with freshly isolated thymocyte for 1 and 3 hours or incubated with thymocyte supernatant (Thy.sup) for 3 hours. Thymocyte supernatant was obtained from thymocytes cocultured with TEC for 12 hours. EMSA were performed with 6 μg of cell extracts probed with the³²P-labeled IL6-κB oligonucleotide. Cytoplasmic proteins were treated with 0.2% deoxycholic acid before binding to dissociate NF-κB/IκB inhibitors. Nuclear complexes contained p50 and p65 subunits, as assessed by the band supershifting obtained with anti-p105 or anti-p65 antisera. DNA binding activity was quantitated by gel densitometry and expressed as a percentage of untreated controls (top of the gels). (B and C) PAGE analysis of NF-IL6 phosphorylation and isoform composition of cytoplasmic and nuclear extracts prepared from¹⁴C-leucine–labeled TEC untreated and cocultured with thymocytes for 1, 3, and 5 hours or incubated with thymocyte supernatants for 5 hours. (B) Immunoblotting of cytoplasmic and nuclear lysate (20 μg) probed with the anti–NF-IL6 antiserum. (C) Immunoblotting of cytoplasmic and nuclear NF-IL6-immunoprecipitates (10³ cpm/lane) probed with antiphosphoserine MoAbs. Arrowheads indicate the positions of proteins with apparent molecular weights of 24, 36, and 43 kD. Sections of gels are shown. The results are representative of at least three experiments performed independently by using TEC and thymocytes derived from different donors.