p ampkα thr 172  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: Acute Simvastatin Inhibits K ATP Channels of Porcine Coronary Artery Myocytes

doi: 10.1371/journal.pone.0066404

Figure Lengend Snippet: Simvastatin (lipophilic) crosses the plasma membrane and reaches the sacroplasmic reticulum (SR) of vascular myocytes. Binding of simvastatin to SR leads to the release of ryanodine (Ryr)-sensitive Ca 2+ into the cytosol. Elevation of Ca 2+ activates CaMK II which leads to the subsequent activation (phosphorylation) of AMPKα. Phosphorylation of AMPKα-Thr 172 causes [glucose] o uptake with the participation of SGLT1 and Na + /K + ATPase. Increase in cytosolic [glucose] leads to an elevation of ATP levels via oxidative phosphorylation. Elevation of [ATP] i serves two purposes: (1) closure of vascular K ATP channels, (2) providing phosphate groups for cellular proteins (e.g. PP2A and AMPK) phosphorylation. Phosphorylation of PP2A occurs downstream of AMPK phosphorylation. PP2A phosphorylation results in PP2A inactivation which “releases” AMPK and thus phosphorylation of AMPKα-Thr 172 resulted. AICAR produces similar effects as simvastatin except the initial step involves LKB1-Ser 428 phosphorylation.

Article Snippet: Non-specific sites were blocked with 5% non-fat dry milk (Bio-Rad, USA) for 120 min, and the blots were then incubated with individual type of antibody: anti-HMG CoA reductase, 1∶1,000 (Upstate Biotechnology, USA); anti-p-HMG CoA reductase-Ser 871 , 1∶1,000 (Kinasource, UK); anti-CYP450 3A4, 1∶1,000 (Affinity Bioreagent, USA); anti-PP2A, 1∶1,000 (Upstate Biotechnology, USA); anti-p-PP2A-Tyr 307 , 1∶1,000 (Upstate Biotechnology, USA); anti-AMPK, 1∶1,000 (Upstate Biotechnology, USA), anti-p-AMPKα-Thr 172 , 1∶1,000 (Upstate Biotechnology, USA); anti-LKB1, 1∶1,000 (Upstate Biotechnology, USA) and anti-p-LKB1-Ser 428 , 1∶1,000 (Upstate Biotechnology, USA) overnight at 4°C.

Techniques: Binding Assay, Activation Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Piperazine ferulate prevents high-glucose-induced filtration barrier injury of glomerular endothelial cells

doi: 10.3892/etm.2021.10607

Figure Lengend Snippet: AMPK mediates HG-induced filtration barrier injury of GEnCs. (A) Structure of PF. (B) Effect of HG on the permeability of GEnCs to FITC-dextran. (C) Western blot analysis of p-AMPKα (Thr-172) and total AMPK. (D) Densitometric analysis of AMPK, p-AMPKα (Thr-172) and the p-AMPKα (Thr-172)/AMPK ratio. (E) Effect of AICAR (1 mM) on the HG-induced permeability of GEnCs. (F) Representative images of glycocalyx staining with FITC-WGA. Data are presented as the mean ± standard deviation of three repeats. ** P<0.01, *** P<0.001 vs. control group; ## P<0.01 vs. HG group. AMPK, adenosine monophosphate-activated protein kinase; HG, high glucose; GEnCs, glomerular endothelial cells; FITC, fluorescein isothiocyanate; p-, phosphorylated-; AICAR, 5-aminoimidizole-4-carboxamide riboside; WGA, wheat germ agglutinin.

Article Snippet: The renal tissue slices were incubated overnight with anti-p-AMPKα (Thr-172) or Hpa-1 antibodies at 4˚C overnight, followed by incubation with the corresponding secondary antibody conjugated with horseradish peroxidase (Boster Biological Technology; cat. no. BM3894; diluted with secondary antibody dilution buffer, 1:1,000) and 3,3'-diaminobenzidine peroxidase substrate.

Techniques: Filtration, Permeability, Western Blot, Staining, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Piperazine ferulate prevents high-glucose-induced filtration barrier injury of glomerular endothelial cells

doi: 10.3892/etm.2021.10607

Figure Lengend Snippet: PF alleviates endothelial glycocalyx injury in vivo . (A) Transmission electron microscopy of glomeruli extracted from different groups of animals. (B) The number of glomerular endothelial fenestrations. (C) FITC-WGA staining for endothelial glycocalyx in the glomerulus. Magnification, x400. (D) Quantification analysis of FITC-WGA staining. (E) Immunohistochemical images of p-AMPKα (Thr-172). Magnification, x400. (F) Quantification analysis of immunohistochemical staining of p-AMPKα (Thr-172). (G) Immunohistochemical staining of Hpa-1. Magnification, x400. (H) Quantification analysis of immunohistochemical staining of Hpa-1. Data are presented as the mean ± standard deviation of three repeats. ** P<0.01, *** P<0.01 vs. the control group; # P<0.05, ## P<0.01 vs. the model group. PF, piperazine ferulate; FITC, fluorescein isothiocyanate; WGA, wheat germ agglutinin; p-, phosphorylated-; AMPK, adenosine monophosphate-activated protein kinase; Hpa-1, heparanase-1.

Article Snippet: The renal tissue slices were incubated overnight with anti-p-AMPKα (Thr-172) or Hpa-1 antibodies at 4˚C overnight, followed by incubation with the corresponding secondary antibody conjugated with horseradish peroxidase (Boster Biological Technology; cat. no. BM3894; diluted with secondary antibody dilution buffer, 1:1,000) and 3,3'-diaminobenzidine peroxidase substrate.

Techniques: In Vivo, Transmission Assay, Electron Microscopy, Staining, Immunohistochemical staining, Standard Deviation