Fig. 2: p53 interacts with SIRT6 in vivo and in vitro in response to palmitic acid (PA) treatment

a, b HCT116 cells were transfected with GFP-SIRT6 and FLAG-p53 plasmids and then exposed to 0.2 mM PA for 18 h. Then, protein was extracted for co-immunoprecipitation (co-IP) using an anti-FLAG or anti-GFP antibody, followed by western blotting using an anti-GFP or anti-FLAG antibody to detect the interaction between p53 and SIRT6. c HCT116 cells were treated with or without 0.2 mM PA for 18 h and the protein was extracted for co-IP using an anti-SIRT6 antibody, followed by western blotting using an anti-SIRT6 or anti-p53 antibody to detect the endogenous interaction between p53 and SIRT6. d His-p53 protein (cloned into pET28b⁺ and expressed in bacteria) was purified in vitro and incubated with GST or a GST-SIRT6 fusion protein (purified from bacteria by using vector pGEX-4T3). Western blotting or Coomassie Brilliant Blue (CBB) staining was performed to detect the direct binding of p53 and SIRT6 in vitro. ^# indicates the specific bands. e Schematic of plasmids encoding full-length (1–355 aa, FL) SIRT6 and SIRT6 fragments (1–34 aa, N terminus; 35–276 aa, core domain; 277–355 aa, C terminus). f GST-SIRT6 FL or fragments were incubated with His-p53, and western blotting with an anti-His antibody or CBB staining was performed to detect the interaction. g Schematic of the plasmids encoding FL p53 and the p53 fragments (1–99 aa, 100–300 aa, 301–393 aa, 301–355 aa and 356–393 aa). h GST-p53 FL or fragments were incubated with His-SIRT6, and analyzed by western blotting with an anti-His antibody or by CBB staining to detect the interaction. IgG immunoglobulin, IB immunoblot; IP immunoprecipitation