Effect of CD28 activation on NFκB activation. (A) IκBα and Bcl-x_L expression. 8226 or U266 cells were cultured with or without anti-CD28 mAb beads as indicated, and analyzed by Western blot using Abs specific for IκBα, Bcl-x_L, or actin as indicated. (B) Nuclear NFκB binding activity. 8226 or U266 cells were cultured with or without immobilized anti-CD28 mAb as indicated, and nuclear extracts analyzed by EMSA for binding to ³²P-labeled primers containing consensus NFκB binding sites. For supershift assays, samples were first incubated with no antibody (−), anti-p50, anti–Rel B, anti–c-Rel, or anti-p65. Data are representative of 3 independent experiments. (C) Binding to/blocking CD86 does not induce NFκB signaling. U266 were treated as in panel B except with or without CD28-Ig (100 μg/mL), and analyzed after 24 hours for nuclear NFκB binding activity. Data are representative of 2 experiments. (D) Up-regulation of Rel B expression by CD28 activation. U266 were treated with control or anti-CD28 mAb (soluble; 1 μg/mL) for 24 hours and analyzed for Rel B and actin expression by Western blot. Data are representative of 2 experiments.