Figure 5: HDAC3 deacetylates MAT IIα.

(a) Overexpression of HDAC3, but not other HDACs, decreases the acetylation level of MAT IIα. Each of the HA-tagged HDAC1-7 was co-transfected with flag-tagged MAT IIα into HEK293T cells and the acetylation levels of MAT IIα were determined by standard western blotting. (b) HDAC3 decreases K81 acetylation of endogenous MAT IIα. HEK293T cells were transfected with HA-HDAC3. MAT IIα K81 acetylation was detected using K81Ac antibody. (c) HDAC3 knockdown decreases MAT IIα protein. HEK293T cells were transfected with siHDAC3 or control. Cells were harvested 48 h after transfection, and lysates were analysed by western blotting. (d) HDAC3 knockdown destabilizes MAT IIα. HEK293T cells were transfected with siHDAC3 or control, respectively. CHX chase treatment was applied as indicated. Cell lysates were directly subjected to western blotting (upper panel) and normalized against β-actin. The lower 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<0.01; ***P<0.001. (e) Folate increases the interaction between HDAC3 and MAT IIα. HEK293T cells were cultured with or without folate for 48 h and treated with MG132 for 6 h before harvest. Interaction between endogenous MAT IIα and HDAC3 was determined by co-IP and western blotting. (f) GST-MAT IIα can readily pull-down P300 and HDAC3. BL21 E. coli transformed with pGEX-GST-MAT IIα plasmid was induced (or not induced) by isopropyl-β-D-thiogalactoside. Protein was then purified through GST antibody-conjugated column and incubated with HEK293T cell lysates (lysed by 0.3% Nonidet P40 buffer) before re-purified through immunoprecipitation and subjected to western blotting.