Fig. 4: The K17 and K86 were essential acetylation sites of ACLY for ARHGEF3 to recruit SIRT2 for deacetylating and stabilizing ACLY.

A Six ACLY acetylation-deficient mutants. B, C H1299 and A549 cells were co-transfected with pcDNA3.1-HA-ARHGEF3, and ACLY mutants, the acetylation of ACLY-K17R and K86R was checked by immunoprecipitation and western blot. D, E H1299 and A549 cells were co-transfected with ARHGEF3 siRNA or shRNA and ACLY mutants, the acetylation of ACLY-K17R and 86 R was checked by immunoprecipitation and western blot. F The interaction between ARHGEF3 and ACLY, ACLY-K17R, 86 R. G The effect of ARHGEF3 overexpression on SIRT2-ACLY(K17R)/(K86R) interaction was detected by western blot. H The effect of ARHGEF3 knockdown on SIRT2-ACLY (K17R)/(K86R) interaction was detected by western blot. I H1299 cells were transfected with Flag-tagged SIRT2 and Flag-tagged ACLY-K17R or 86 R and HA-tagged ARHGEF3. The acetylation of ACLY-K17R and 86R were determined by western blot. J Flag-tagged SIRT2, Flag-tagged ACLY-K17R or 86R and ARHGEF3 siRNA were co-transfected in H1299 cells. Western blot was used to check the acetylation of ACLY-K17R and 86R. K, L Cycloheximide (CHX) (25 μg/ml) was incubated for the indicated period with H1299 cells transfected with ACLY mutants and pcDNA3.1-HA-ARHGEF3 or ARHGEF3 siRNA. Western blot was used to detect the effects of ARHGEF3 on the degradation rates of ACLY-K17R and 86 R (left). Degradation curve ACLY-K17R and 86 R (right). The results represent the average of three independently repeated experiments (mean ± SD), ns no significance.