FIGURE 2.

TIP60 interacts with and acetylates ThPOK. A, HEK 293T cells were co-transfected with expression vectors encoding for HA-ThPOK or FLAG-TIP60, as indicated. Cell lysates were immunoprecipitated (IP) with either anti-FLAG or anti-HA antibody and detected by Western blotting (IB). B, nuclear extracts from CD4⁺ T cells were immunoprecipitated with anti-TIP60 or control IgG antibodies and then analyzed by Western blotting. C, TIP60 acetylates ThPOK. HEK 293T cells were co-transfected with HA-ThPOK and increasing amounts of FLAG-TIP60; 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. D, CD4⁺ T cells were transduced with lentivirus containing shRNA sequences targeting CK (control) or TIP60. Cells were cultured with anti-CD3/CD28 dynal beads and selected with puromycin for 2 days. Selected cells were lysed, and cell lysates were immunoprecipitated with anti-ThPOK antibody and analyzed by Western blotting. E, resting CD4⁺ T cells were stimulated with α-CD3 and α-CD28 antibodies for the indicated time periods. Cells were lysed with RIPA buffer; cell lysates were immunoprecipitated with anti-ThPOK antibody and analyzed by Western blotting.