Figure 1: MAT IIα is acetylated at lysine 81.
From: Acetylation of MAT IIα represses tumour cell growth and is decreased in human hepatocellular cancer
(a) Exogenous MAT IIα is acetylated. Flag-MAT IIα was transfected into HEK293T and Chang’s cells followed by TSA treatment (10 μM) for 18 h. Flag-MAT IIα acetylation was detected by anti-acetyllysine (Pan-Ac) antibody. (b) Alignment of MAT IIα lysine 81 and adjacent protein sequence from different organisms. Hs: Homo Sapient (human); Pt: Pan troglodytes (chimpanzee); Cl: Canis lupus familiaris (dog); Bt: Bos taurus (bull); Mm: Mus musculus (mouse); Rn: Rattus norvegicus (Norway rat); Dm: Drosophila melanogaster (fruit fly). (c) TSA increases acetylation level of wild-type MAT IIα but not mutants. Ectopically expressed wild-type MAT IIα, K81R and K81Q were transfected into HEK293T cells followed by TSA treatment, MAT IIα acetylation was analysed by western blotting. (d) Specificity of K81 site-specific acetylation antibody was determined by dot blotting assay. (e) MAT2A knockdown confirmed the K81Ac antibody detection. HEK293T cells were transfected with MAT2A siRNA or scramble control and cell lysates were subjected to western blotting. Signal detected by K81Ac antibody decreased as MAT IIα was knocked down. (f) K81Ac antibody is specific to MAT IIα K81 acetylation. HEK293T cells were lysed and directly subjected to western blotting. The four repetitively loaded lanes were clipped into four membranes and were exposed to antibodies as indicated, separately. For peptide block, acetylated K81 peptide (the antigen we used for K81Ac antibody generation) or non-acetylated K81 peptide (mimicking MAT IIα protein) were added to the diluted K81Ac antibodies (1:200) in a final concentration of 300 μg ml-1, cultivated at 37 °C for 1 h before applying to membranes. Four clipped membranes were aligned and exposed together when developing film to make sure their relative signal intensities are comparable. (g) TSA promotes MAT IIα acetylation at K81 in different cell lines. HEK293T, Hela and HepG2 cells were treated as indicated. K81 acetylation was detected by K81Ac antibody and normalized against MAT IIα protein.
