Figure 4: P300 acetylates MAT IIα.

(a) Overexpression of P300, not other HATs, increases acetylation of exogenous MAT IIα. HEK293T cells were transfected as indicated and acetylation of flag-MAT IIα was determined using Pan-Ac antibody. (b) Overexpression of P300 increases K81 acetylation of endogenous MAT IIα. HEK293T cells were transfected with flag-P300 and K81 acetylation of MAT IIα was determined using K81Ac antibody. (c) P300 knockdown decreases MAT IIα K81 acetylation. HEK293T cells were transfected with siP300 or control and MAT IIα acetylation levels were determined by western blotting. (d) P300 decreases protein levels of MAT IIα. Flag-P300 was transfected in declined doses into HEK293T cells and cell lysates were measured by western blotting. (e) P300 knockdown increases MAT IIα stability. HEK293T cells were transfected with siP300 or control and cultured under folate-deprived condition before CHX was added and treated for indicated durations. Levels of endogenous MAT IIα protein were determined by western blotting and normalized against β-actin (left panel). The right panel showcases relative protein amounts of different groups. Error bars represent ±s.d. of triplicate experiments. The two-tailed Student’s t-test was used. *P<0.05; **P<0.01; ***P<0.001. (f) Folate-deprivation enhances MAT IIα–P300 interaction. HEK293T cells were transfected with flag-tagged MAT IIα and cultured in normal or folate-deprived conditions. The interaction between flag-MAT IIα and endogenous P300 was determined by co-IP followed by western blotting. (g) P300 acetylates MAT IIα at K81 in vitro. Ectopically expressed GST-MAT IIα from BL21 E. coli was purified through GST antibody-conjugated beads and incubated with P300 (immunoprecipitation-purified and eluted). K81 acetylation levels were analysed using K81Ac antibody and normalized against GST-MAT IIα.