Immunoblot Assay

Spleens from OT-I-transferred, DC±IL-2c-treated mice were harvested on D6, stained with anti-Thy1.1-APC antibody (Clone OX-7, BD Pharmingen) and purified with anti-APC-antibody magnetic beads according to standard AutoMacs protocols. A total of 2 × 10⁶ OT-I cells were washed with cold PBS and lysed in NP40 buffer (20 mM HEPES [pH 7.9], 100 mM NaCl, 5 mM EDTA, 0.5 mM CaCl, 1% Nonidet P-40, 1 mM PMSF, 10 μg/ml leupeptin, 5 μg/ml aprotinin, and 1 mM Na₃VO₄). Ten micrograms of protein were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with Bim or β-actin antibodies (Cell Signal Technology; Beverly, MA) as indicated. Antibodies were detected with goat anti-rabbit conjugated to horseradish peroxidase (Santa Cruz) and Supersignal (Thermo Scientific). Images were captured with ImageJ software.