Fig. 4

Sirt2 acts as a physiological RRM2 deacetylase. a siRNA screening strategy to identify RRM2 deacetylase. b H1299 cells were transfected with a library of siRNAs targeting 11 HDAC family members or a library of siRNAs targeting seven sirtuin family members, followed by IP using RRM2 antibody, and western blot with Ac-K antibody. c H1299 cells were co-transfected with HA-RRM2 along with Flag-tagged Sirt1-7, followed by co-IP with agarose-conjugated Flag antibody and western blot to detect the Flag-tagged Sirt(s)-associated HA-RRM2. d Immunostaining using RRM2 and Sirt2 antibodies in H1299 cells. Scale bar: 25 μm. e Co-IP was carried out using RRM2 antibody to detect RRM2/Sirt2 interaction in H1299 cells. f H1299 cells were co-transfected with HA-RRM2 along with Flag-tagged WT or deacetylase-inactive (H187Y) Sirt2, followed by analysis of HA-RRM2 acetylation. g Sirt2 was depleted from H1299 cells using Sirt2 shRNA1 or 2, followed by analysis of RRM2 acetylation. h RRM2 acetylation was analyzed following treatment of H1299 cells with Sirt2 inhibitor AGK2. i HA-RRM2 was isolated from H1299 cells treated with TSA/NAM and then incubated with Flag-WT or H187Y inactive mutant Sirt2 protein purified from 293T cells, followed by analysis of HA-RRM2 acetylation. j Sirt2 was depleted from H1299 cells using Sirt2 shRNA1 or 2, followed by analysis of RNR activity. The error bars indicate ± s.d. of three separate experiments. **P <0.01, by two-tailed t test