Journal: PLoS ONE

Article Title: Green Tea Extract Rich in Epigallocatechin-3-Gallate Prevents Fatty Liver by AMPK Activation via LKB1 in Mice Fed a High-Fat Diet

doi: 10.1371/journal.pone.0141227

Figure Lengend Snippet: Western blotting analysis of protein expression in the liver on different experimental groups of AMPK—LKB1 pathway (A) pLKB1, (B) LKB1, (C) pAMPKα, (D) AMPKα 1/2. Image shows demonstrative bands of the analyzed proteins and respective housekeeping protein (β-tubulin) in liver. Data are expressed in mean ± s.e.m. * p<0.05 Control diet and EGCG (CE) group versus Control diet and Water (CW) group. # p<0.05 High-fat diet and EGCG (HFE) group versus HFW group. $ p<0.05 HFW versus CW.

Article Snippet: The membranes were blocked in 1% bovine serum albumin overnight at room temperature, and then incubated overnight with the following primary antibodies: pAMPK α1/2 (sc-33524), AMPK α1/2 (sc-25792), ACCα (sc-30212), FAS (sc-20140), SREBP-1 (sc-8984), ChREBP (sc-21189), SIRT-1 (sc-15404) purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Control

Journal: Journal of molecular neuroscience : MN

Article Title: Effects of Intracerebroventricular Glycogen Phosphorylase Inhibitor CP-316,819 Infusion on Hypothalamic Glycogen Content and Metabolic Neuron AMPK Activity and Neurotransmitter Expression in Male Rat

doi: 10.1007/s12031-019-01471-0

Figure Lengend Snippet: Summary of graded intracerebroventricular ( icv ) CP-319,819 (CP) dosage effects on ventromedial hypothalamic nucleus (VMN) glycogen/glucose content and metabolic neuron 5′-AMP-activated kinase (AMPK) activity and neurotransmitter protein expression during eu- versus hypoglycemia

Article Snippet: Membranes were blocked (2 h) with TBS containing 0.1% Tween-20 and 2.0% bovine serum albumin prior (36–48 h; 4 °C) incubation in a Next Advance Blotbot with rabbit primary antisera against AMPK α1⁄2 (prod. no. 2532, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), pAMPK α1⁄2 (prod. no. 2531, 1:1000; Cell Signal.

Techniques: Activity Assay, Expressing

Journal: Journal of molecular neuroscience : MN

Article Title: Effects of Intracerebroventricular Glycogen Phosphorylase Inhibitor CP-316,819 Infusion on Hypothalamic Glycogen Content and Metabolic Neuron AMPK Activity and Neurotransmitter Expression in Male Rat

doi: 10.1007/s12031-019-01471-0

Figure Lengend Snippet: Effects of CP pretreatment on IIH patterns of glutamate decarboxylase65/67 (GAD65/67), Fos, 5′-adenosine monophosphate-activated protein kinase (AMPK), and phosphoAMPK (pAMPK) protein expression in VMN GABAergic neurons. GAD65/67-immunoreactive neurons located in the right hemi-hypothalamic VMN were laser-catapult microdissected for stain-free Western blot analysis of GAD65/67 (panel a), Fos (panel b), AMPK (panel c), and pAMPK (panel d) protein expression in V- or CP-pretreated eu- versus hypoglycemic male rats. For each protein of interest, triplicate pools of n = 50 heat-denatured cell lysates were created within each treatment group. Target protein band optical densities (O.D.) were detected and quantified in a Bio-Rad ChemiDoc MP Imaging System equipped with Image Lab™ software; target protein O.D. values were normalized to total protein content of individual lanes. Data depict mean normalized protein O.D. values + S.E.M. for groups of animals pretreated by icv V or CP (2.5 or 10.0 mg/24 h) infusion prior to sc INS or V injection. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Membranes were blocked (2 h) with TBS containing 0.1% Tween-20 and 2.0% bovine serum albumin prior (36–48 h; 4 °C) incubation in a Next Advance Blotbot with rabbit primary antisera against AMPK α1⁄2 (prod. no. 2532, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), pAMPK α1⁄2 (prod. no. 2531, 1:1000; Cell Signal.

Techniques: Expressing, Staining, Western Blot, Imaging, Software, Injection

Journal: Journal of molecular neuroscience : MN

Article Title: Effects of Intracerebroventricular Glycogen Phosphorylase Inhibitor CP-316,819 Infusion on Hypothalamic Glycogen Content and Metabolic Neuron AMPK Activity and Neurotransmitter Expression in Male Rat

doi: 10.1007/s12031-019-01471-0

Figure Lengend Snippet: Hypoglycemic patterns of steroidogenic factor-1 (SF-1), Fos, 5′-AMPK, and pAMPK protein expression in male rat VMN SF-1 neurons; impact of graded CP dosage pretreatment. Laser-catapult microdissected VMN SF-1-immunoreactive neurons were evaluated by stain-free Western blotting to determine effects of CP on SF-1 (panel a), Fos (panel b), AMPK (panel c), and pAMPK (panel d) protein responses to IIH. Data depict mean normalized protein O.D. values + S.E.M. for V/V (solid white bars), V/I (diagonal-striped white bars), CP-2.5/V (solid light gray bars), CP-2.5/INS (diagonal-striped light gray bars), CP-10.0/V (solid dark gray bars), and CP-10.0/INS (diagonal-striped dark gray bars) treatment groups. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Membranes were blocked (2 h) with TBS containing 0.1% Tween-20 and 2.0% bovine serum albumin prior (36–48 h; 4 °C) incubation in a Next Advance Blotbot with rabbit primary antisera against AMPK α1⁄2 (prod. no. 2532, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), pAMPK α1⁄2 (prod. no. 2531, 1:1000; Cell Signal.

Techniques: Expressing, Staining, Western Blot

Journal: Journal of molecular neuroscience : MN

Article Title: Effects of Intracerebroventricular Glycogen Phosphorylase Inhibitor CP-316,819 Infusion on Hypothalamic Glycogen Content and Metabolic Neuron AMPK Activity and Neurotransmitter Expression in Male Rat

doi: 10.1007/s12031-019-01471-0

Figure Lengend Snippet: Effects of icv CP infusion on VMN nitrergic nerve cell neuron nitric oxide synthase (nNOS), Fos, 5′-AMPK, and pAMPK protein responses to IIH. VMN nNOS-immunoreactive neurons were laser-microdissected for stain-free Western blot analysis of nNOS (panel a), Fos (panel b), AMPK (panel c), and pAMPK (panel d) protein expression. Data depict mean normalized protein O.D. values + S.E.M. for the following treatment groups: V/V, V/I, CP-2.5/V, CP-2.5/INS, CP-10.0/V, and CP-10.0/INS. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Membranes were blocked (2 h) with TBS containing 0.1% Tween-20 and 2.0% bovine serum albumin prior (36–48 h; 4 °C) incubation in a Next Advance Blotbot with rabbit primary antisera against AMPK α1⁄2 (prod. no. 2532, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), pAMPK α1⁄2 (prod. no. 2531, 1:1000; Cell Signal.

Techniques: Staining, Western Blot, Expressing

Journal: The Journal of Neuroscience

Article Title: Structural and Functional Rescue of Chronic Metabolically Stressed Optic Nerves through Respiration

doi: 10.1523/JNEUROSCI.3652-17.2018

Figure Lengend Snippet: Low lactate level accompanies AMPK activation and limits mitochondrial biogenesis and metabolic cofactor pools. A, l-lactate levels in 3-, 6-, and 10-month-old D2G and D2 ONs (n = 8 ONs/group). B, Ratio of pAMPK to AMPK protein in 3-, 6-, and 10-month-old D2G and D2 ONs (n = 8 ONs/group). C, D, Phosphorylated AMPK immunofluorescence (magenta) and GFAP (green) micrographs (C) of human control and glaucoma ONs (n = 4 sections/ON, 2 ONs/group); and 3-month-old D2, and 10-month-old D2G and D2 mice (n = 3 sections/ON, 6 ONs/group; D). Arrows indicate colocalization of pAMPK and GFAP. E, F, Percentage of mean fluorescence intensity in the ROI for pAMPK 3-month-old D2, and 10-month-old D2G and D2 mice (E) and humans (F). G–I, Analyses of NAD+/NADH, CK activity, and PGC1-α levels in 3-, 6-, and 10-month-old D2G and D2 mice. G, NAD+ normalized to NADH levels (n = 6 ONs/group). See Figure 3-1. H, Creatine kinase activity normalized to total protein (n = 6 ONs/group). I, PGC1-α protein levels normalized to total protein levels and then to 3-month-old D2G protein levels (n = 8 ONs/group). All values are presented as the mean ± SEM, one-way ANOVA and Tukey's post hoc test. A, F(5,42) = 22.04, *p = 0.0124; B, F(5,42) = 35.51, **p = 0.0032, ***p = 0.0001; E, F(2,45) = 208.4, ***p = 0.0001; F, t(14) = 14.79, ***p = 0.0001, two-tailed unpaired t test; G, F(5,30) = 5.061, *p = 0.0012; H, F(5,48) = 38.28, **p = 0.0025; I F(5,42) = 5.43, *p = 0.0231. Scale bar, C, D, 20 μm.

Article Snippet: pAMPK α1 Thr 172 , 1:100 for IHC, 1:50 for Wes , AB_11032916 , Novus Biologicals , Rabbit.

Techniques: Activation Assay, Immunofluorescence, Control, Fluorescence, Activity Assay, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Structural and Functional Rescue of Chronic Metabolically Stressed Optic Nerves through Respiration

doi: 10.1523/JNEUROSCI.3652-17.2018

Figure Lengend Snippet: Monocarboxylate transport changes and AMPK activation occur with acute glaucoma injury. A, CTB (green) after intraocular injection from retina to SC of control and bead injected Mito-CFP mice. Dark regions of the superficial, retinorecipient areas of the SC indicate lack of axon transport from the retina. See Figure 4-1. B, Percentage area fraction of CTB transport in SC (n = 10 per group). C, RGCs immunolabeled for the RGC-specific antigen RBPMS in control and bead-injected Mito-CFP mice. D, Quantification of RGC density in control and bead-injected mice (n = 16 control mice; n = 14 bead-injected mice). E–G, Protein analysis by capillary electrophoresis of pAMPK/AMPK (E), MCT1 (F), and MCT2 (G). Proteins normalized to total protein (n = 6 ONs/group). All values are presented as the mean ± SEM, *two-tailed unpaired t test. B, t(16) = 15.07, ***p = 0.0001; D, t(27) = 5.412, ***p = 0.0001; E, t(10) = 4.165, ***p = 0.0019; F, t(10) = 3.114, *p = 0.0110; G, t(10) = 6.536, **p = 0.00110. Scale bars: A, 100 μm; C, 50 μm.

Article Snippet: pAMPK α1 Thr 172 , 1:100 for IHC, 1:50 for Wes , AB_11032916 , Novus Biologicals , Rabbit.

Techniques: Activation Assay, Injection, Control, Immunolabeling, Electrophoresis, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Structural and Functional Rescue of Chronic Metabolically Stressed Optic Nerves through Respiration

doi: 10.1523/JNEUROSCI.3652-17.2018

Figure Lengend Snippet: List of antibodies used for IHC and capillary electrophoresis analyses

Article Snippet: pAMPK α1 Thr 172 , 1:100 for IHC, 1:50 for Wes , AB_11032916 , Novus Biologicals , Rabbit.

Techniques: Electrophoresis

Journal: ASN NEURO

Article Title: Norepinephrine Regulation of Adrenergic Receptor Expression, 5’ AMP-Activated Protein Kinase Activity, and Glycogen Metabolism and Mass in Male Versus Female Hypothalamic Primary Astrocyte Cultures

doi: 10.1177/1759091420974134

Figure Lengend Snippet: Patterns of Hypothalamic Astrocyte 5’-AMP-Activated Protein Kinase (AMPK) and PhosphoAMPK (pAMPK) Protein Expression in Male and Female Rats: Effects of NE Stimulation. Data depict mean normalized AMPK or pAMPK protein O.D. measures ± S.E.M. for vehicle- (white bars) or NE-treated (gray bars) male and female cultured astrocytes. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: ; RRID: AB_330331); phosphoAMPK α1/2 (pAMPK; 1:1,000, prod. no. 2535 L; Cell Signal.

Techniques: Expressing, Cell Culture

Journal: Scientific Reports

Article Title: Metformin prevents stroke damage in non-diabetic female mice with chronic kidney disease

doi: 10.1038/s41598-021-86905-9

Figure Lengend Snippet: Metformin pre-conditioning enhances adenosine monophosphate-activated protein kinase (AMPK) activation and reduces canonical NFκB activation in CKD mice. ( A ) Representative images of phosho-AMPK, IΚBα, phospho-P65, phospho-ACC (Ser-79) and total-ACC western blots performed on SHAM and CKD mice exposed, or not, to metformin. ( B ) Quantitative data showing higher AMPK phosphorylation in ischemic hemispheres of CKD-metformin than CKD-vehicle treated mice. ( C ) Quantitative data showing higher ACC phosphorylation in ischemic hemispheres of CKD-metformin than CKD-vehicle treated mice. ( D ) Quantitative data showing higher IΚBα expression in ischemic hemispheres of CKD-metformin than CKD-vehicle treated mice. ( E ) Quantitative data showing lower P65 phosphorylation in ischemic hemispheres of CKD-metformin than CKD-vehicle treated mice. Results are expressed as the median, interquartile, and min–max and show data from at least 6 animals per group. Statistical analysis was performed using a non-parametric Mann–Whitney test. *p < 0.05, **p < 0.01, CKD-vehicle vs. SHAM-vehicle mice. $ p < 0.05, $$ p < 0.01, CKD-metformin vs. CKD-vehicle mice.

Article Snippet: Western blotting was performed using rabbit polyclonal anti-phosphorylated AMPK α1/2 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-phosphorylated ACC (ser-79) (1:1000, Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-ACC (1:1000, Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phosphorylated NFκB p65 (1:1000, Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-IƘBα (1:1000, Cell Signaling Technology, Danvers, MA), or rabbit polyclonal anti-LC3A/B (1:1000, Cell Signaling Technology, Danvers, MA) as described previously .

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Expressing, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Metformin prevents stroke damage in non-diabetic female mice with chronic kidney disease

doi: 10.1038/s41598-021-86905-9

Figure Lengend Snippet: Hypothetical scheme of the mechanisms by which metformin prevent ischemic stroke damage in non-diabetic mice with CKD. In brain ischemic lesions of non-diabetic CKD mice, AMPK activity, which is known to block microglia/macrophages M1 polarization, is impaired. The recruited microglia/macrophages consequently display increased M1 and reduced M2 polarization. The subsequent inflammation may be responsible for the elevated apoptosis, increased infarct volumes and poorer functional outcomes. A 5-week course of pre-conditioning with metformin rescued the activation of AMPK in mice with CKD. This effect is associated with a significant decrease of macrophage/microglia transition towards the M 1 phenotype, as shown by the decreased expression of the M 1 markers CD16, CD32, and CD86. Metformin-induced AMPK activation blocks NFκB activation and the subsequent release of pro-inflammatory cytokines such as IL-1β. This decrease in inflammation may be responsible for less brain-cell apoptosis, thus reducing the cortical infarct volume and subsequent neurobehavioral disorders.

Article Snippet: Western blotting was performed using rabbit polyclonal anti-phosphorylated AMPK α1/2 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-phosphorylated ACC (ser-79) (1:1000, Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-ACC (1:1000, Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phosphorylated NFκB p65 (1:1000, Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-IƘBα (1:1000, Cell Signaling Technology, Danvers, MA), or rabbit polyclonal anti-LC3A/B (1:1000, Cell Signaling Technology, Danvers, MA) as described previously .

Techniques: Activity Assay, Blocking Assay, Functional Assay, Activation Assay, Expressing