Anti p ampk

Manufactured by Cell Signaling Technology

Sourced in United States, China

Anti-p-AMPK is an antibody product from Cell Signaling Technology. It is designed to detect phosphorylated AMPK, a key cellular energy sensor.

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P-AMPK (404 citations) Anti-AMPK (214 citations) Anti-p-AMPK T172 (9 citations) Anti-p-AMPK antibody (5 citations) Rabbit-anti-p-AMPK (T172) (4 citations) Rabbit anti–p-AMPK (Thr172) (3 citations) Anti-P-AMPK (#2535) (2 citations) Anti-phosphorylated AMPK (p-AMPK; (2 citations) Anti-phosphorylated (p)AMPK (2 citations) Anti-rabbit p-AMPK (thr 172 (2 citations)

103 protocols using anti p ampk

Gene Expression and Protein Analysis

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Total RNA was isolated and purified using TRIzol reagent (Invitrogen, #15596026) according to the manufacturer's protocol. cDNA was prepared to employ a CycleScript RT premix (Bioneer, #K-2044-CFG). All primer sequences are listed in Table 1. Expression levels were calculated using a SensiFAST SYBR Lo-ROX Kit (Bioline, #BIO-94020) and a commercial detection system (BioRad, #CFX96).
Total proteins were extracted into RIPA buffer (Thermo Fisher Scientific, #89900) containing a phosphatase inhibitor (Sigma-Aldrich, #4906845001) and a protease inhibitor (Roche, #43693159001), subjected to 10–16% (w/v) Tris-glycine SDS-PAGE, transferred to PVDF membranes using Iblot 2 NC ministacks (Invitrogen, #IB23002), and the membranes blocked with 5% (w/v) skim milk. The primary antibodies were anti-pAMPK (Cell Signaling Technology, #2531S), anti-AMPK (Cell Signaling Technology, #5831S), anti-pACC (Cell Signaling Technology, #3661S), anti-ACC (Cell Signaling Technology, #3662S), and anti-GAPDH (Cell Signaling Technology, #2118S) diluted 1:1000 (primary antibodies) or 1:5000 (secondary antibodies) in TBST (Biosesang, #HT2007) containing 5% (w/v) skim milk. The membranes were then incubated with a peroxidase-conjugated anti-rabbit secondary antibody and signals quantitated using the Immobilion Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500).

Yoon K.J., Park S., Kwak S.H, & Moon H.Y. (2021). Effects of Voluntary Running Wheel Exercise-Induced Extracellular Vesicles on Anxiety. Frontiers in Molecular Neuroscience, 14, 665800.

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Immunoblotting for Autophagy Markers

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The primary antibodies used were the anti‐Ubiquitin (Santa Cruz Biotechnology, Inc., sc‐8017), anti‐foxo (COSMO BIO CO, CAC‐THU‐A‐DFOXO), anti‐26S proteasome α (Santa Cruz Biotechnology, Inc., sc‐65,755), anti‐26S Proteasome p54 (Rpn10) (Santa Cruz Biotechnology, Inc., sc‐65,746), anti‐pAMPK (Cell Signaling Technology, #2535), anti‐GABARAP (Atg8a) (Cell Signaling Technology, #13733), anti‐Lamp1 (Abcam, ab30687), anti‐ATP5A (Abcam, ab14748), anti‐Actin (Cell Signaling Technology, #8457), and anti‐Gapdh (Sigma‐Aldrich, G9545). The anti‐ref(2)P/p62 antibody was kindly donated from Prof. G. Juhász. The secondary antibodies Peroxidase AffiniPure Donkey anti‐Mouse IgG (715‐035‐150) and Peroxidase AffiniPure Donkey anti‐Rabbit IgG (711‐035‐152) were purchased from Jackson ImmunoResearch Laboratories, Inc. The anti‐Rabbit‐IgG Alexa Fluor 647 (711‐605‐152), anti‐Rabbit‐IgG Alexa Fluor 488 (111‐545‐003), anti‐Mouse‐IgG Rhodamine (TRITC) AffiniPure (715‐025‐151), anti‐Mouse‐IgG Alexa Fluor 488 (115‐545‐003), and anti‐Mouse IgG DyLight™ 405 (715‐475‐151) antibodies were also from Jackson ImmunoResearch Laboratories, Inc. Ponceau S solution (6226–79) was from Sigma‐Aldrich.

Papanagnou E., Gumeni S., Sklirou A.D., Rafeletou A., Terpos E., Keklikoglou K., Kastritis E., Stamatelopoulos K., Sykiotis G.P., Dimopoulos M.A, & Trougakos I.P. (2022). Autophagy activation can partially rescue proteasome dysfunction‐mediated cardiac toxicity. Aging Cell, 21(11), e13715.

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Western Blot Analysis of Autophagy Markers

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Mouse tissue extracts were prepared by homogenizing tissues in lysis buffer containing 50 mM Tris (pH 7.9), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, proteinase inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany), and Halt phosphatase inhibitor cocktail (Roche Applied Sciences), and then subjected to Western blot analysis with anti-LC3 (1:500, Sigma-Aldrich), anti-P62 (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-Bnip3 (1:500, Cell Signaling Technology), anti-AMPK (1:500, Cell Signaling Technology), anti-p-AMPK (1:500, Cell Signaling Technology), anti-Beclin1 (1:500, Cell Signaling Technology), and anti-GAPDH (1:3,000, Cell Signaling Technology) antibodies. Activation of AMPK was expressed as the ratio of phosphorylated AMPK (pAMPK) to total AMPK (tAMPK).

Zhang D., Lee J.H., Kwak S.E., Shin H.E., Zhang Y., Moon H.Y., Shin D.M., Seong J.K., Tang L, & Song W. (2019). Effect of a Single Bout of Exercise on Autophagy Regulation in Skeletal Muscle of High-Fat High-Sucrose Diet-Fed Mice. Journal of Obesity & Metabolic Syndrome, 28(3), 175-185.

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Baicalin Modulates TLR4/PI3K/AMPK Pathway

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Baicalin (purity ≥ 98%) was obtained from Xi’an Kai Lai Biological Engineering Co., Ltd. (Xi’an, China). Fluoxetine hydrochloride (Flu) was purchased from Changzhou Siyao Pharmaceuticals Co., Ltd. (Changzhou, China). Lipopolysaccharide (LPS), 4,6-diamidino-2-phenylindole (DAPI), and compound C were bought from Sigma-Aldrich Co (St. Louis, USA). TAK-242 (a TLR4 antagonist) and LY294002 (a PI3K inhibitor) were products purchased from Apex Bio (Houston, USA). Poly-d-lysine was obtained from Sigma (MO, USA). Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were supplied by Elabscience Biotechnology Co., Ltd. (Wuhan, China). The antibodies were obtained from the cited commercial sources: anti-p-PI3K (#4228), anti-PI3K (#4292), anti-p-Akt (Ser473, #9271), anti-Akt (#9272), anti-β-actin (#4967), anti-PCNA (#13110), anti-p-AMPK (#2531), and anti-AMPK (#2603) were from Cell Signaling Technology (Beverly, MA, USA); anti-PTEN (sc-7974) and anti-p-PTEN (sc-377573) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-TLR4 (AF7017), anti-p-GSK3β (AF2016), and anti-GSK3β (AF7814) were from Affinity Biosciences (Changzhou, China); and anti-FoxO1 (ab52857), anti-p-FoxO1 (Ser 256, ab131339), and goat anti-rabbit IgG H&L (Alexa Fluor® 488, ab150077) were from Abcam (Cambridge, MA, USA).

Guo L.T., Wang S.Q., Su J., Xu L.X., Ji Z.Y., Zhang R.Y., Zhao Q.W., Ma Z.Q., Deng X.Y, & Ma S.P. (2019). Baicalin ameliorates neuroinflammation-induced depressive-like behavior through inhibition of toll-like receptor 4 expression via the PI3K/AKT/FoxO1 pathway. Journal of Neuroinflammation, 16, 95.

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Kidney Cortex Protein Analysis

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The tissue was harvested from the kidney cortex of the sacrificed animals. Protein was extracted for western blotting from the cortex. The primary antibodies used in this study were Fatty Acid and Lipid Metabolism Antibody Sampler Kit (#8335), anti-TNFR1 (#13377), anti-AMPK (#5832), and anti-p-AMPK (#2535), from Cell Signaling Technology (Danvers, MA, USA); anti-SREBP-1 (sc-13551) and anti-SREBP-2 (sc-13552), from Santa Cruz Biotechnology (Dallas, TX, USA); anti-CD36 (NB400-144ss), anti-β actin (NB600-501), and anti-α tubulin (NB100-690), from Novus Biologicals (Littleton, CO, USA); anti-PPARα (GTX01098), anti-HDAC1 (GTX100513), and anti-histone H3 (GTX122148), from GeneTex (Irvine, CA, USA); and anti-KIM 1 (ab190696, Abcam, Cambridge, UK). The membrane was then subjected to the secondary antibody, either anti-mouse IgG or anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA), which was dissolved in 5% skim milk in TBST for 1 h. Next, the membrane was incubated for 1–2 min in enhanced chemiluminescence mixture (JT96-K004M, T-Pro Biotechnology, Zhonghe, New Taipei City, Taiwan) for visualization. The western blotting assay was repeated at least three times.

Lin Y.C., Wang J.C., Wu M.S., Lin Y.F., Chen C.R., Chen C.Y., Chen K.C, & Peng C.C. (2020). Nifedipine Exacerbates Lipogenesis in the Kidney via KIM-1, CD36, and SREBP Upregulation: Implications from an Animal Model for Human Study. International Journal of Molecular Sciences, 21(12), 4359.

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Multimodal Cancer Treatment Investigation

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The 5-fluorouracil, metformin, and ICG-001 used were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Selleckchem (Houston, TX, USA) respectively. The antibodies, such as anti-PARP, anti-cleaved PARP, anti-caspase 3, anti-cleaved caspase 3, anti-LC3B, anti-ATG9A, anti-CD44, anti-β-catenin, anti-OCT4, anti-β-actin, anti-p-AMPK, and anti-AMPK were procured from Cell Signaling Technology (Danvers, MA, USA). APC conjugated anti-CD44 was procured from BioLegend (San Diego, CA, USA). All the primers were procured from Integrated DNA Technologies (San Diego, CA, USA).

Roy S., Zhao Y., Yuan Y.C, & Goel A. (2022). Metformin and ICG-001 Act Synergistically to Abrogate Cancer Stem Cells-Mediated Chemoresistance in Colorectal Cancer by Promoting Apoptosis and Autophagy. Cancers, 14(5), 1281.

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Western Blot Analysis of Hippocampal Proteins

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For Western blot analysis, total protein was isolated from the hippocampus and its concentration was determined by Bradford method. The concentration of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was 12%. The gel was transferred to a polyvinylidene fluoride (PVDF) membrane and the filters were blocked for 1 h in 5% non-fat dry milk in TBS-T (25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20). Blots were incubated at 4°C overnight with antibodies as follows: anti-C/EBP-homologous protein (anti-CHOP) (Cell Signaling; 1:1000), anti glucose-regulated protein 78 kD (anti-GRP78) (Proteintech; 1:1000), anti-inhibitor of NF-κB (anti-IκB) (Cell Signaling; 1:1000), anti-p65 (Proteintech; 1:800), anti-AMPK (Cell Signaling; 1:2000), anti-pAMPK (Cell Signaling; 1:2000), anti-SIRT1 (abcam; 1:1000), anti-β-actin (Proteintech; 1:4000). After washing, the membranes were then probed with horseradish peroxidase-conjugated secondary antibody. The bands were then detected by a chemiluminescence detection system. β-actin was used as an internal standard for all of the gels.

Liao D., Chen Y., Guo Y., Wang C., Liu N., Gong Q., Fu Y., Fu Y., Cao L., Yao D, & Jiang P. (2020). Salvianolic Acid B Improves Chronic Mild Stress-Induced Depressive Behaviors in Rats: Involvement of AMPK/SIRT1 Signaling Pathway. Journal of Inflammation Research, 13, 195-206.

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Cell Viability and Apoptosis Assays

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Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Bovine Serum Albumin (BSA) and Trypsin (0.5% EDTA) were obtained from GIBCO^TM, (Invitrogen Corporation). Artemisinin, Penicillin/Streptomycin, Lipofectamine^R 2000 reagent (Invitrogen Co.,CA, USA), DMSO, H₂O₂ were obtained from Sigma Aldrich (St. Louis, MO, USA). Sodium Azide (NaN₃) were obtained from Acros Organic, (New Jersey, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Cell ROXs Deep Red Reagent, and Hoechst 33342 were purchased from Molecular Probes (Eugene, or, USA). Pierce BCA protein assay kit and Halt^TM Protease and phosphatase inhibitor cocktail were purchased from Thermo Scientific USA, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanineiodide (JC-1) from Beyotime, Annexin V-FITC/PI were purchased from BBI Life Sciences. anti-p-AMPK, anti-AMPK and anti-β-Actin antibodies were purchased from Cell Signaling Technology (CST) (Beverly, MA, USA). Anti-Rabbit IgG HRP-conjugated secondary antibody was purchased from Promega (Madison, Wl, USA).

Li S., chaudhary S.C., Zhao X., Gaur U., Fang J., Yan F, & Zheng W. (2019). Artemisinin Protects Human Retinal Pigmented Epithelial Cells Against Hydrogen Peroxide-induced Oxidative Damage by Enhancing the Activation of AMP-active Protein Kinase. International Journal of Biological Sciences, 15(9), 2016-2028.

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Isolation and Western Blot Analysis of ARC

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Ad libitum-fed or 24 h-fasted mice were deeply anesthetized with isoflurane before removing the brain. ARC was isolated by microdissection and solubilized in lysis buffer (38.5 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, 1% Triton-X 100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, and 250 mM sucrose) with protease inhibitor on ice. Protein concentration was determined by bicinchoninic acid assay and the same amount of protein was loaded on polyacrylamide gels for SDS separation. Western blot was performed using either anti-V5 (Cell Signaling Technology, Rabbit mAb #13202) or anti-pAMPK (Cell Signaling Technology Rabbit mAb #2535).

Srisai D., Yin T.C., Lee A.A., Rouault A.A., Pearson N.A., Grobe J.L, & Sebag J.A. (2017). MRAP2 regulates ghrelin receptor signaling and hunger sensing. Nature Communications, 8, 713.

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Western Blot Analysis of Cardiac Proteins

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Heart tissue was homogenized in RIPA (Millipore, Billerica MA) lysis buffer and centrifuged at 12,000 g for 20 min at 4°C. Samples containing equal amounts of protein were separated on a 10 or 12% SDS-polyacrylamide gel in a mini-gel apparatus (Mini-PROTEAN II, Bio-Rad). Proteins were then transferred to nitrocellulose membranes and subsequently blocked with 5% BSA in tris buffered saline (TBS)-tween 20. Membranes were incubated overnight at 4°C in primary antibodies: anti-PTEN (Cell Signaling, Cat#9552), anti-pAMPK (T172, Cell Signaling, Cat#2535), anti-AMPK (Cell Signaling, Cat#2532), anti-p62 (Enzo Life Sciences, Cat#GP62-C), anti-LC3B (Cell Signaling, Cat#3868S), anti-GAPDH (Cell Signaling, Cat#2118L), and anti-Pink1 (Abcam, Cat#ab23707). Blots were washed with TBS-Tween 20 and incubated with horseradish peroxidase (HRP) conjugated secondary antibody. Membranes were subsequently washed and exposed by enzymatic chemiluminescence in a GelDoc system (Bio-Rad) [25 (link)].

Roe N.D., Xu X., Kandadi M.R., Hu N., Pang J., Weiser-Evans M.C, & Ren J. (2014). Targeted Deletion of PTEN in Cardiomyocytes Renders Cardiac Contractile Dysfunction through Interruption of Pink1-AMPK Signaling and Autophagy. Biochimica et biophysica acta, 1852(2), 290-298.

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