Journal: Animal bioscience

Article Title: Guanidinoacetic acid regulated postmortem muscle glycolysis associated with AMPK signaling and protein acetylation.

doi: 10.5713/ab.24.0418

Figure Lengend Snippet: Figure 4. Effect of guanidinoacetic acid administration on protein abundances of LKB1 (A), 539

Article Snippet: The primary 143 antibodies were phosphor liver kinase B1 (LKB1, Thr189, no. 3054s), phosphor-AMKPα (Thr172, 144 no. 2531s), rabbit acetylated-lysine antibody (no. 9441) and glyceraldehyde-3-phosphate 145 dehydrogenase (GAPDH, no. 97166), and all primary antibody were purchased from Cell Signaling 146 Technology Inc. (Beverly, MA, United States) and diluted at 1:1000.

Techniques:

Journal: Redox biology

Article Title: Fenofibrate improves vascular endothelial function and contractility in diabetic mice.

doi: 10.1016/j.redox.2018.09.024

Figure Lengend Snippet: Fig. 4. Endothelial function improvement by fenofibrate is through normalizing eNOS activity in diabetic mice. A, Protein expression of PPARα in aortas from vehicle and fenofibrate-treated control mice (Con) and diabetic mice (DM). B, Protein expression of LKB1 and phosphorylations of LKB1 in nucleus and cytosol of aortas from vehicle and fenofibrate-treated Con and DM. Phosphorylation of AMPKα (C) and eNOS (D) in aortas from vehicle and fenofibrate-treated Con and DM. ***p < 0.001 vs Con, ###p < 0.001 vs vehicle-treated DM. n = 4.

Article Snippet: Membranes were blocked by 5% fat-free milk powder and immunodetected with specific primary antibodies to β-actin, eNOS, p- LKB1 (Ser334), LKB1, p-AMPK (Thr172), AMPK, COX-2, NF-κB p65 (Ser 468) (Abcam, Cambridge, UK); p-eNOS (Ser1177) (Cell Signaling Technology, Beverly, MA, USA), Histone H3 (Beyotime Biotechnology, Shanghai, China) and second horseradish peroxidase-labeled IgG antirabbit (or mouse) antibody (Beyotime Biotechnology, Shanghai, China).

Techniques: Activity Assay, Expressing, Control, Phospho-proteomics

Journal: Cardiovascular Research

Article Title: Cardiomyocyte-specific deletion of Sirt1 gene sensitizes myocardium to ischaemia and reperfusion injury

doi: 10.1093/cvr/cvy033

Figure Lengend Snippet: Impaired ischemic AMPK activation in aged and icSIRT1 KO hearts. (A) Immunoblotting measured the phosphorylation level of AMPK of ischemic region in response to different time points of myocardial ischaemia in young SIRT1flox/floxand young icSIRT1 KO hearts. Values are means ± SEM, n = 4, *P < 0.05 vs. sham, respectively; †P < 0.05 vs. SIRT1flox/flox ischaemia using 2-way ANOVA with Tukey’s post-test. (B) Immunoblotting of phosphorylation level of AMPK and AMPK downstream acetyl CoA carboxylase (ACC) in SIRT1flox/floxand icSIRT1 KO hearts under sham or 10 min of ischaemia. Values are means ± SEM, n = 3–4, *P < 0.05 vs. sham, respectively; †P < 0.05 vs. SIRT1flox/flox ischaemia using 2-way ANOVA with Tukey’s post-test. (C) Immunoblotting of phosphorylation level of AMPK and AMPK downstream ACC in the ischemic region of young and aged hearts under sham or 10 min of ischaemia. Values are means ± SEM, n = 4–6, *P < 0.05 vs. sham, respectively; †P < 0.05 vs. young ischaemia using 2-way ANOVA with Tukey’s post-test. (D) Immunoblotting showed the phosphorylation levels of AMPK upstream kinase LKB1 in the ischemic region of young, aged, and icSIRT1 KO hearts under sham or ischaemia conditions, the immunoprecipitation of LKB1 were used for assessing acetylation and ubiquitination levels of LKB1 occurred in young, aged, and icSIRT1 KO hearts as shown by the immunoblotting with antibodies recognized acetyl-lysine and ubiquitin, respectively.

Article Snippet: Mouse LKB1 antibody was obtained from Novus Biologicals (Littleton, CO).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Immunoprecipitation, Ubiquitin Proteomics

Journal: Cardiovascular Research

Article Title: Cardiomyocyte-specific deletion of Sirt1 gene sensitizes myocardium to ischaemia and reperfusion injury

doi: 10.1093/cvr/cvy033

Figure Lengend Snippet: Hyper-acetylation of LKB1 attenuates AMPK signalling and GLUT4 translocation in icSIRT1 KO hearts. (A) The immunoblotting of immunoprecipitated LKB1 with antibody recognized acetyl-lysine showed that myocardial ischaemia triggered deacetylation of LKB1 in SIRT1flox/floxbut not in icSIRT1 KO hearts. (B) The immunoprecipitation of a scaffold protein Sestrin2 showed the association between Sestrin2 and AMPKα was abolished in icSIRT1 KO hearts, LKB1 immunoprecipitation data also showed the diminished interaction between LKB1 and Sestrin2 in the icSIRT1 KO hearts vs. SIRT1flox/floxhearts. (C) Photolabelling with BGPA to measure the cell surface GLUT4 in SIRT1flox/floxand icSIRT1 KO hearts under sham or ischaemia conditions. Value are means ± SEM, n = 4 per group, *P < 0.05 vs. sham, respectively, †P < 0.05 vs. WT ischaemia using 2-way ANOVA with Tukey’s post-test. (D) The immunoprecipitation of GLUT4 showed a GLUT4 anchor protein TUG (Tether containing UBX domain for GLUT4) was dissociated with GLUT4 in the SIRT1flox/floxhearts in response to ischemic stress, while the interaction between GLUT4 and TUG was augmented in icSIRT1 KO hearts during ischaemia. Value are means ± SEM, n = 4–5 per group, *P < 0.05 vs. sham, respectively; †P < 0.05 vs. SIRT1flox/floxischaemia using 2-way ANOVA with Tukey’s post-test. (E) Upper: The immunoblotting of cardiac damage marker Troponin I, oxidative stress marker Shc phosphorylation, autophagic influx proteins eEF2, ULK1, LC3II/LC3I, and p62, inflammation-related signalling p65 modifications, and mitochondrial proteins associated with apoptosis BNIP3, Bcl-2, Bcl-xL, and Bax, to show the sensitivity of SIRT1flox/floxand icSIRT1 KO hearts to ischemic stress; Lower: quantitative analysis of the relative levels of proteins in SIRT1flox/floxand icSIRT1 KO hearts under sham or ischaemia conditions. Values are means ± SEM, n = 3–6, *P < 0.05 vs. sham, respectively; †P < 0.05 vs. SIRT1flox/floxischaemia using 2-way ANOVA with Tukey’s post-test.

Article Snippet: Mouse LKB1 antibody was obtained from Novus Biologicals (Littleton, CO).

Techniques: Translocation Assay, Western Blot, Immunoprecipitation, Marker, Phospho-proteomics