FIGURE 3.

TIP60-mediated acetylation of lysine 360 stabilizes ThPOK. A, HA-ThPOK was co-transfected with increasing amounts of FLAG-TIP60 into HEK 293T cells; cell lysates were analyzed by Western blotting (IB). B, HA-ThPOK was expressed in HEK 293T cells in the presence or absence of FLAG-TIP60. Cells were treated with 20 μg/ml cycloheximide (CHX) for the indicated periods. Levels of ThPOK were determined by Western blotting. C, CD4⁺ T cells were transduced with lentivirus containing shRNA sequences targeting CK (control), TIP60, or ThPOK. Cells were cultured with anti-CD3/CD28 dynal beads and selected with puromycin for 5 days. Selected cells were lysed for the detection of ThPOK and TIP60 levels by Western blotting. D, domain structure of ThPOK protein. The potential acetylated lysines by TIP60 were labeled as indicated. E, FLAG-TIP60 was expressed in HEK 293T cells with HA-tagged ThPOK or its deletion variants. Cell lysates were immunoprecipitated (IP) with anti-HA antibody and detected by WB with the indicated antibodies. F, FLAG-TIP60 was expressed in HEK 293T cells with HA-tagged ThPOK, its lysine mutants, or deletion variants. Cell lysates were analyzed by Western blotting. G, HEK 293T cells were transfected with HA-tagged ThPOK, its lysine mutants, or FLAG-TIP60 as indicated. 1 mm nicotinamide and 50 μm Ex-527 were added 6 h before cell harvesting. Cell lysates were immunoprecipitated with anti-HA antibody and detected by Western blotting with the indicated antibodies.