rabbit anti human ampkα1  (Cell Signaling Technology Inc)

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: α-ALA Attenuates Cardiac Injury in Diabetic Heart Disease Mice by Modulating Inflammation and AMPKα1

doi: 10.2147/DMSO.S537517

Figure Lengend Snippet: Effect of α-ALA on AMPKα1 in HDF +STZ induced diabetic mice. ( A ) Relative mRNA expression of AMPKα1; ( B ) Western blot analyses of AMPKα1 and p-AMPKα1; ( C ) AMPKα1 protein quantifications; ( D ) p-AMPKα1 protein quantifications. Control: Normal control mice; DHD: HDF +STZ; α-ALA-L: HDF +STZ mice treated with 2 g/kg of α-ALA; α-ALA-H: HDF +STZ mice treated with 4 g/kg of α-ALA. Data are expressed as mean ± SD, n ≥5. # P < 0.05, ## P < 0.01 is compared with the Control group. *P < 0.05, **P < 0.01 is compared with the DHD group.

Article Snippet: The membranes were incubated overnight with primary antibodies specific for AMPKα1 (1:2000, 10,929-2-AP, Proteintech, China), p-AMPKα1 (1:500, AF3423, Affinity, China), and β-actin (1:10,000, 81,115-1-RR, Proteintech, China), followed by horseradish peroxidase-conjugated secondary antibodies.

Techniques: Expressing, Western Blot, Control

Journal: Cancer Discovery

Article Title: Aged and BRCA -Mutated Stromal Cells Drive Epithelial Cell Transformation

doi: 10.1158/2159-8290.CD-24-0805

Figure Lengend Snippet: Aging-associated AMPK derepression of the JNK/c-JUN/WT1 axis results in the hrMSC protumorigenic phenotype. A, DNA methylation in nMSCs, hrMSCs, and CA-MSCs at the promoter region of the PRKAG1 gene. B, qRT-PCR expression data of AMPK-related genes in nMSC vs. hrMSC. C, Hypothesized AMPK/JNK/c-JUN/WT1 axis. D – F, Characterization of phospho-AMPKα1 and total AMPKα1, JNK, and c-JUN in nMSCs and hrMSCs. Representative Western blots are shown with corresponding densitometries. Patient samples were grouped, and statistical significance was determined using the Student’s t test. For both nMSCs and hrMSCs, N > 3 patients. G and H, Simple linear regressions correlating p-JNK and p-c-JUN with WT1 expression on a per-sample basis via densitometry. I, Quantification of AMPK Western blot levels in nMSC vs. hrMSC at passage 4 vs. passage 8. J, Western blot of hrMSCs treated with increasing doses of the JNK inhibitor SP600125. Quantified band intensities are shown. K and L, Western blot and quantification of AMPKα1 and pAMPKα1 following BC1618 treatment for 24 hours. M, BC1618-treated hrMSCs were analyzed by flow cytometry for MDA fluorescence. P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparison analysis. M – P, Quantification of p-JNK, p-c-JUN, and WT1 bands following treatment of hrMSCs with either BC1618 or SP600125. Q, Representative 20× images of FTE that were cocultured at a 1:1 ratio with hrMSC AMPKα1-m-GFP or empty vector (EV) transduced. The percentage of cells with >9 53BP1 foci was quantified by fluorescence microscopy. More than 3 fields per condition were taken to analyze 150–200 individual cells. The Student’s t test was used to determine the significance of Q . R, Aging-associated loss of AMPK phosphorylation and expression results in the derepression of JNK phosphorylation, resulting in increased WT1 expression, oxidative stress, and FTE DNA DSBs.

Article Snippet: Membranes were blocked for 1 hour at RT using TBS Licor Buffer (927-60001) with 0.01% Tween 20 and then stained using rabbit anti-human pAMPKα1 (Cell Signaling Technology, Cat# 2535, RRID:AB_331250; 1:500), rabbit anti-human AMPKα1 (Cell Signaling Technology, Cat# 2532, RRID:AB_330331; 1:500), rabbit anti-human WT1 (Abcam, Cat# ab89901, RRID:AB_2043201; 1:1,000), rabbit anti-human p-JNK T182/183 (Cell Signaling Technology, Cat# 9251, RRID:AB_331659; 1:1,000), rabbit anti-human JNK (Cell Signaling Technology, Cat# 9252, RRID:AB_2250373; 1:1,000), rabbit anti-human p-c-JUN S63 (Cell Signaling Technology, Cat# 2361, RRID:AB_490908; 1:1,000), rabbit anti-human c-JUN (Cell Signaling Technology, Cat# 9165, RRID:AB_2130165; 1:1,000), rabbit anti-human vinculin (Cell Signaling Technology, Cat# 4650, RRID:AB_10559207; 1:1,000), rabbit anti-human GAPDH (Cell Signaling Technology, Cat# 2118, RRID:AB_561053; 1:5,000), and rabbit anti-human β-actin (Abcam, Cat# ab8227, RRID:AB_2305186; 1:2,000) overnight, rocking, at 4°C.

Techniques: DNA Methylation Assay, Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Fluorescence, Comparison, Plasmid Preparation, Microscopy, Phospho-proteomics

Journal: Experimental & molecular medicine

Article Title: Neurotensin inhibits AMPK activity and concurrently enhances FABP1 expression in small intestinal epithelial cells associated with obesity and aging.

doi: 10.1038/s12276-025-01461-w

Figure Lengend Snippet: Fig. 2 NTS deficiency reverses HFD-inhibited p-AMPK expression. a–c Male mice were fed either a LFD or a HFD for 6 weeks at weaning. BW was measured before mice were euthanized (a). Protein was extracted from jejunal mucosal scrapings and analyzed by western blot, and data show representative results from five mice per group (b). Densitometric analysis of p-AMPKα was performed by ImageJ and normalized by the average of AMPKα1/α2 (c). n = 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. d Images of jejunal monolayers were taken by inverted microscope at d1 and d2. e IF of jejunal monolayers at d2 and counterstained with Hoechst and phalloidin. f IF of jejunal monolayers at d2 using anti-AMPKα1 antibody (red) and counterstained with Hoechst (blue). g Western blot of proteins isolated from jejunal monolayers from male WT mice fed a LFD or HFD for 6 weeks at weaning; cells were treated with or without NTS (10 nM) for 10 min. Densitometric analysis of p- AMPKα was performed by ImageJ and normalized by AMPKα. n = 4 mice per group. h Male WT mice (4 months old) were maintained on NC. Western blot analysis of protein isolated from jejunal monolayers treated with NTS (10 nM) or PA (30 μM) alone or in combination for 24 h. Densitometric analysis of p-AMPKα was performed by ImageJ and normalized by AMPKα. n = 4 mice per group. *P < 0.05, **P < 0.01. i Male WT mice were fed a LFD or HFD for 6 weeks at weaning. Jejunal crypts were isolated for monolayer culture; cells were treated with AICAR at different concentrations for 24 h. n = 3 mice per group.

Article Snippet: AMPKα1 (sc19128), AMPKα2 (sc19129), FABP1 (sc-374537) and FABP2 (sc-374482) antibodies were from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Inverted Microscopy, Isolation