NF-κB binding activity and NF-IL6 phosphorylation in TEC untreated and treated with anti-3, -6, -β1, and -β4 MoAbs in the presence or absence of cross-linking. (A) NF-κB binding activity of nuclear extracts (6 μg) prepared from TEC untreated or treated for 3 hours with the various MoAbs at 5 μg/mL. When indicated, bound MoAbs were cross-linked by the addition of F(ab)₂ GAM at 10 μg/mL for 2 hours. (B and C) PAGE analysis of NF-IL6 phosphorylation and isoform composition in nuclear extracts prepared from ¹⁴C-leucine–labeled TEC untreated or treated as previously mentioned. (B) Immunoblotting of nuclear lysate (20 μg) probed with anti–NF-IL6 antiserum. (C) Immunoblotting of nuclear NF-IL6 immunoprecipitates (10³ cpm/lane) probed with antiphosphoserine MoAbs. Arrowheads indicate the positions of proteins with apparent molecular weight of 36 and 43 kD. Sections of gels are shown. Results shown are representative of three experiments performed independently by using TEC and thymocytes derived from different donors.