FIG. 6.

NF-κB is necessary but not sufficient for costimulation-independent proliferation. (A) EMSA for NF-κB DNA-binding activities. CD4⁺ thymocytes (2 × 10⁵ cells per well) were stimulated with indicated concentrations of plate-bound anti-CD3ɛ with or without soluble anti-CD28 for 24 h. Nuclear extracts were prepared from the stimulated and unstimulated thymocytes and used in EMSAs. The indicated subunits present in each of the specific NF-κB binding complexes were identified by supershift assays (data not shown). A ubiquitously expressed nonspecific binding complex (NS) served as a loading control. (B) Inhibition of CD4⁺ thymocyte proliferation by an inhibitor of NF-κB activation. Proliferation cultures were set up as described for Fig. 5B, except the inhibitor peptide (NBD) or control peptide (NBA) were included at the concentration of 200 μM. [³H]thymidine incorporation was measured as described for Fig. 4A.