Fig. 2. AMPK primes SIRT7 phosphorylation by GSK3β.

a, b Immunoblots showing the interaction of endogenous SIRT7 and GSK3β by immunoprecipitation of the indicated proteins. c, d Immunoblotting and related quantification (n = 3 biologically independent samples) of SIRT7 pan p-S/T levels in HEK293 cells transfected with increasing amounts of GSK3β plasmids with or without LiCl (10 mM) treatment; error bars: means ± SEM; P values were calculated by one-tailed Student’s t-test. e SIRT7 and the indicated mutants were overexpressed in HEK293 cells and then extracted for immunoblotting analysis of phosphorylation levels with an anti-pan-p-S/T antibody. The relative intensity measured by Image J® and is shown the individual bands. f Immunoblotting analysis of the sequential immunoprecipitations based on anti-HA or anti-FLAG antibodies in HEK293 cells co-expressing ectopic HA-SIRT7 and Flag-AMPK. Arrow indicates the specific band for FLAG-AMPK. g Immunoblots showing the binding of endogenous SIRT7 to GSK3β in HEK293 cells under glucose starvation (1 h) or Compound C treatment (CC, 5 µM). h Immunoblots showing the binding of WT-SIRT7 or T263A to GSK3β in HEK293 cells transfected with the indicated plasmids. i Immunoblotting analysis of SIRT7 phosphorylation levels after in vitro kinase assays based on active GSK3β and AMPK complexes immunoprecipitated from AICAR-treated HEK293 cells. SIRT7 phosphorylation was detected by probing with an anti-pan p-S/T antibody. j, k Immunoblots showing SIRT7 phosphorylation levels in HEK293 cells treated as indicated. The anti-p-SIRT7 antibody was generated by immunization with a Thr255/Ser259 phosphorylated peptide. Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.