Figure 2. T-bet undergoes proteasome-dependent degradation during mitosis.

(A) Quantification of T-bet signal in interphase vs. metaphase T cells represented in Figure 1A. T-bet signal was compared between interphase and metaphase blasts imaged in the same field of view (n=61). Error bars indicate SEM. (B) CD4⁺ T cells were activated in vitro for 24h and then synchronized with nocodazole. Cells were washed free of drug, cultured in vitro with or without the proteasome inhibitors MG-132, calpain inhibitor I, or lactacystin. Cell lysates were prepared at 0, 15, or 30m after nocodazole washout. T-bet and β-actin levels were assessed by immunoblotting. (C) CD4⁺ or CD8⁺ T cells were prepared as in (B) and T-bet levels assessed by intracellular staining at 0 and 30m after nocodazole washout. T-bet levels of naïve T cells and activated cells not treated with nocodazole (“freely cycling”) are also shown. Results are representative of three separate experiments.