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. 2014 Nov 10;124(12):5453–5465. doi: 10.1172/JCI76611

Figure 2. SIRT2 deacetylates and activates ALDH1A1.

Figure 2

(A) SIRT2, not SIRT1, decreased K353 acetylation and increased ALDH1A1 activity. 293T cells were transfected with the indicated plasmids. FLAG-ALDH1A1 was immunoprecipitated, and ALDH1A1 activity was assayed. (B) SIRT2 deacetylase activity was required to increase ALDH1A1 activity. 293T cells were cotransfected with FLAG-ALDH1A1 and SIRT2 WT or the inactive mutant H187Y. (C) SIRT2 knockdown increased K353 acetylation and decreased ALDH1A1 activity. 293T cells were transfected with the indicated plasmids and SIRT2 siRNA oligonucleotides. ALDH1A1 was immunoprecipitated and activity was assayed. ALDH1A1 acetylation at K353 was determined by Western blotting. (A–C) ALDH1A1 activity was normalized to protein levels, and relative enzyme activity data represent the mean ± SD of triplicate experiments. (D) SIRT2 overexpression decreased endogenous ALDH1A1 K353 acetylation. Plasmid expressing SIRT2 WT or the inactive mutant H187Y was transfected into HEPG2 cells, and endogenous K353 acetylation and ALDH1A1 were determined. (E) SIRT2 knockdown increased endogenous ALDH1A1 K353 acetylation. siRNA oligonucleotide targeting SIRT2 was transfected into HEPG2 cells, and the levels of endogenous K353 acetylation, ALDH1A1, and SIRT2 protein were determined by Western blotting. (F and G) ALDH1A1 bound to SIRT2. (F) The indicated plasmids were cotransfected into 293T cells, and ALDH1A1-SIRT2 binding was determined by IP and Western blotting. (G) Interaction of endogenous ALDH1A1 and SIRT2 in HEPG2 cells was determined by IP and Western blotting.