Journal: Acta biochimica et biophysica Sinica

Article Title: PGAM5 deacetylation mediated by SIRT2 facilitates lipid metabolism and liver cancer proliferation.

doi: 10.3724/abbs.2023155

Figure Lengend Snippet: Figure 2. SIRT2 mediates PGAM5-K191 deacetylation (A) Flag-tagged PGAM5 was stably expressed in PLC cells treated with TSA and/or NAM, followed by immunoprecipitation using an anti-Flag antibody and western blot analysis of the lysine acetylation (K-Ac) of PGAM5. (B) Localization of sirtuin family deacetylases in cells. (C) Immunoprecipitation was performed using anti-Flag antibody or IgG in PLC cells. Flag-PGAM5 and HA- SIRT2 expression were determined by western blot analysis. (D) PLC cells stably expressing Flag-PGAM5 were further transfected with vectors expressing EV or HA-SIRT2. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. The acetylation of Flag-PGAM5 was detected by western blot analysis. (E) PLC cells stably expressing Flag-PGAM5 were further transfected with vectors expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. The acetylation of Flag-PGAM5 was determined by western blot analysis. (F) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with vectors expressing Flag-PGAM5- WT, Flag-PGAM5-K191R and Flag-PGAM5-K191Q. The above cells were subsequently infected with viruses expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. The acetylation of Flag-PGAM5 was detected by western blot analysis. (G) Diagram of the mechanism by which SIRT2 regulates the deacetylation of PGAM5-K191.

Article Snippet: Primary antibodies against the following proteins were used: PGAM5 (Cat# sc-515880; Santa Cruz, Santa Cruz, USA), PGAM5 (Cat# 28445-1-AP; Proteintech, Rosemont, USA), SIRT2 (Cat# 19655-1-AP; Proteintech), SIRT2 (Cat# sc-28298; Santa Cruz), ME1 (Cat# 16619-1-AP; Proteintech), acetylated-lysine (Cat# 9441; CST, Beverly, USA), acetylated-lysine (Cat# PTM-105; Jingjie PTM BioLab, Hangzhou, China), phosphorylation-serine (Cat# sc-81514; Santa Cruz), HA-HRP (Cat# 2999; CST), HA (Cat# 51064-2-AP; Proteintech), mouse IgG1 isotype control (Cat# 66360-1-Ig; Proteintech), Flag (Cat# F1804; Sigma-Aldrich), and β-actin (Cat# 66009-1-Ig; Proteintech).

Techniques: Stable Transfection, Immunoprecipitation, Western Blot, Expressing, Transfection, shRNA, Knockdown, Infection

Journal: Acta biochimica et biophysica Sinica

Article Title: PGAM5 deacetylation mediated by SIRT2 facilitates lipid metabolism and liver cancer proliferation.

doi: 10.3724/abbs.2023155

Figure Lengend Snippet: Figure 3. The deacetylation of PGAM5-K191 mediated by SIRT2 facilitates ME1 dephosphorylation and activity (A) Flag-PGAM5 and HA-ME1 were co-overexpressed in PLC cells. Immunoprecipitation was performed using an anti-Flag antibody or IgG, followed by western blot analysis of HA-ME1 and Flag-PGAM5. (B) PLC cells stably expressing Flag-PGAM5 were further transfected with vectors expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. ME1 and Flag-PGAM5 were determined by western blot analysis. (C) PLC cells stably expressing Flag-PGAM5 were further transfected with empty vector (EV) or vectors expressing HA-SIRT2. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. ME1 and Flag-PGAM5 were determined by western blot analysis. (D) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with EV or vectors expressing Flag-wild-type- PGAM5, Flag-PGAM5-K191R and Flag-PGAM5-K191Q. The above cells were subsequently infected with viruses expressing HA-ME1. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. Flag-PGAM5 and HA-ME1 were determined by western blot analysis. (E) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with EV or vectors expressing HA-PGAM5, HA-PGAM5-K191R and HA-PGAM5-K191Q. The above cells were knocked down with ME1 shRNA and were subsequently infected with viruses expressing Flag-ME1-WT or Flag-ME1-S336A. Immunoprecipitation was performed using an anti-Flag antibody or IgG in PLC cells. Phosphorylation of ME1, Flag-ME1 and HA-PGAM5 was determined by western blot analysis. (F) PLC cells with stable knockdown of endogenous PGAM5 were further transfected with vectors expressing PGAM5-WT, PGAM5-K191R and PGAM5-K191Q. The above cells were knocked down with ME1 shRNA and were subsequently infected with viruses expressing Flag-ME1-WT or Flag-ME1-S336A. Meanwhile, the cells were infected with the viruses expressing NTC or SIRT2 shRNA. Immunoprecipitation was performed using an anti-Flag antibody or IgG in the above cells. Phosphorylation of ME1, Flag-ME1, HA-PGAM5 and SIRT2 was detected by western blot analysis. (G) The metabolic enzyme activity of ME1 was detected by a kit assay in the PLC cells used in (F). The enzyme activity data are expressed as the mean±SD (n=3) and compared with the corresponding groups. *P<0.05, ns, no significant difference.

Article Snippet: Primary antibodies against the following proteins were used: PGAM5 (Cat# sc-515880; Santa Cruz, Santa Cruz, USA), PGAM5 (Cat# 28445-1-AP; Proteintech, Rosemont, USA), SIRT2 (Cat# 19655-1-AP; Proteintech), SIRT2 (Cat# sc-28298; Santa Cruz), ME1 (Cat# 16619-1-AP; Proteintech), acetylated-lysine (Cat# 9441; CST, Beverly, USA), acetylated-lysine (Cat# PTM-105; Jingjie PTM BioLab, Hangzhou, China), phosphorylation-serine (Cat# sc-81514; Santa Cruz), HA-HRP (Cat# 2999; CST), HA (Cat# 51064-2-AP; Proteintech), mouse IgG1 isotype control (Cat# 66360-1-Ig; Proteintech), Flag (Cat# F1804; Sigma-Aldrich), and β-actin (Cat# 66009-1-Ig; Proteintech).

Techniques: De-Phosphorylation Assay, Activity Assay, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Transfection, shRNA, Plasmid Preparation, Knockdown, Infection, Phospho-proteomics