Inhibitors of lysosomal degradation and GSK3β increase KLF13 expression in resting human T cells. (A) Resting human T lymphocytes were treated with MG-132 (2.5 or 5 μg/mL), chloroquine (CQ; 50nM or 100nM), or CMA (0.5 or 1 μg/mL) for 4 hours, and the cell lysates were subjected to Western blot analysis. (B) Human T lymphocytes activated by anti-CD3 and CD28 Ab for 5 days were treated with MG-132 (5 μg/mL), CQ (100nM), or CMA (1 μg/mL) for 4 hours, and the cell lysates were subjected to Western blot analysis. (C) Resting T lymphocytes were treated with MG-132 (1 μg/mL), GSK3 inhibitors (1nM for GSK3 and 0.2mM for GSK3β), or p38 inhibitor (1 μg/mL) for 4 hours, and the cell lysates were subjected to Western blot analysis. (D) Resting human T cells were transfected with nontargeting control (NTC) or GSK3β-depleting siRNA. Cells were harvested after 24 hours, and cell lysates were assayed for GSK3β and KLF13 by Western blot analysis. (E) Recombinant GSK3β was incubated with recombinant GST or GST-KLF13 in the presence of [γ-³²P]ATP. The protein kinase reaction products were resolved by SDS-PAGE, and phosphorylation was detected by autoradiography. (F) ³⁵S-in vitro–translated KLF13 was incubated at 30°C in a ubiquitination reaction mix containing SCF(Fbw7γ) purified from HEK293T and/or recombinant GSK3β as indicated. Reactions were stopped by adding SDS loading buffer at the indicated times, resolved by SDS-PAGE, and analyzed by autoradiography.