Ampkα

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China, Germany, Japan

AMPKα is a catalytic subunit of the AMP-activated protein kinase (AMPK) complex. AMPK is a cellular energy sensor that plays a crucial role in maintaining energy homeostasis within cells. AMPKα is responsible for the phosphorylation and activation of AMPK, which then regulates various metabolic pathways in response to changes in cellular energy levels.

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Anti-p-AMPKα(Thr172) Total AMPKα T-AMPKα Rabbit polyclonal anti-AMPKα Anti-p-AMPKα Phospho-AMPKα (p-AMPKα, Rabbit anti-phospho-AMPKα (Thr172) Rabbit anti-phospho-AMPKα (Thr172) (2535 Anti-phosphor-AMPKα Phosphorylated AMPKα (Thr172)

280 protocols using ampkα

Adipocyte Differentiation Protocol

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Mouse embryo fibroblast 3T3-L1 (ATCC^®CL173) and normal human primary subcutaneous preadipocytes (ATCC^® PCS210010) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Dulbecco’s modified Eagle’s medium high glucose (DMEM), penicillin/streptomycin and L-glutamine were purchased from Mediatech, Inc. (Manassas, VA). PPARγ (Cat# 2443S), C/EBPα (Cat# 2295S), FAS (Cat# 4233S), HMGB2 (Cat# 14163S), AMPKα (Cat# 5832S) and phospho(p)-AMPKα (Cat# 50081S-Thr172), Akt1(Cat# 75692S) and p-Akt1 (Cat# 9018S-Ser473) antibodies were from Cell Signaling Technology (Boston, MA). GAPDH (Cat# G9545) antibodies were from Sigma-Aldrich (St. Louis, MO). All secondary antibodies (Cat# 305-035-045) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All other chemicals were obtained from Sigma-Aldrich unless otherwise stated.

LEUNG I., VEISAGA M.L., ESPINAL M., ZHANG W., BARNUM R, & BARBIERI M.A. (2021). Anti-lipid droplets accumulation effect of Annona montana (mountain soursop) leaves extract on differentiation of preadipocytes. Biocell : official journal of the Sociedades Latinoamericanas de Microscopia Electronica ... et. al, 46(3), 567-578.

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Western Blot Analysis of Brain Proteins

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Sagittally cut hemi-brains brains were homogenized in ice-cold RIPA buffer (PBS, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) with freshly added protease inhibitors (Protease Inhibitor Cocktail P8340 and, Phosphatase Inhibitor Cocktail 1&2, Sigma-Aldrich, St. Louis, MO; Phenylmethylsulfonylfluoride (PMSF), Fluka-Biochemica, Switzerland), incubated on ice for 30 min and centrifuged at 14,000 g for 10 min at 4°C. After centrifugation, the supernatant was collected and total protein concentrations were measured using the Bradford method. Tissue lysate was added to electrophoresis sample buffer and separated by SDS/PAGE under reducing conditions on a 12% separation gel. Proteins were transferred to nitrocellulose membranes using the iBlot dry-blotting system (Invitrogen Corp., Carlsbad, CA). Unspecific binding was blocked by incubation in 5% milk blocking buffer (PBS, 5% nonfat milk and 0.1% Tween 20). Membrane bound proteins were immunoblotted with antibodies to BDNF, NGF, NT3, Nrf2 (Santa Cruz Biotech, Santa Cruz, CA), p-AMPK, AMPKα (Cell Signaling, Beverly, MA), p-Nrf2 (BIOSS Antibodies, Woburn, MA) and β-actin (Abcam Inc., Cambridge, MA). Signals were developed using ECL reagent (Amersham Pharmacia Biotech, Buckinghamshire, England). Densities of the bands were evaluated using Image J software.

Allard J.S., Perez E.J., Fukui K., Carpenter P., Ingram D.K, & de Cabo R. (2015). Prolonged metformin treatment leads to reduced transcription of Nrf2 and neurotrophic factors without cognitive impairment in older C57BL/6J mice. Behavioural brain research, 301, 1-9.

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Western Blot Analysis of AMPK Signaling

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Cells were lysed in 50 mM Tris pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 150 mM NaCl, 1 mM Na₃VO₄, 20 mM NaF, 1mM PMSF, 2 μg ml⁻¹ aprotinin, 2 μg ml⁻¹ leupeptin and 0.7 μg ml⁻¹ pepstatin. Protein concentrations of cell extracts were determined by the Bradford assay. Western blot analysis was carried out as previously described (Thai, Thaker et al. 2015 (link)). The following antibodies were used as probes: Actin (Abcam; 1:1000), Phospho-AMPKα Thr172 (Cell Signaling #2535; 1:1000), AMPKα (Cell Signaling #2532; 1:1000), Caspase 3 (Cell Signaling #9662; 1:1000), Cleaved Caspase 3 (Asp175) 5A1E (Cell Signaling #9664; 1:1000), pACC1 (Ser79) D7D11 (Cell Signaling #11818; 1:1000), ACC1 (Cell Signaling #4190; 1:1000).

Thaker S.K., Chapa T., Garcia G J.r., Gong D., Schmid E.W., Arumugaswami V., Sun R, & Christofk H.R. (2019). Differential metabolic reprogramming by Zika virus promotes cell death in human versus mosquito cells. Cell metabolism, 29(5), 1206-1216.e4.

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Immunoblotting Analysis of Autophagy Signaling

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Cells were placed on ice, washed twice with cold PBS and lysed with NETN buffer [50 mM Tris-HCI (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40] plus protease inhibitors. 30–50 μg protein of each sample was used for analysis by immunoblotting. Quantification of western blot films was performed with Image J, with the measure of controls set as 1. Antibodies used include: BRUCE (Bethyl, A300-367A), LC3B (Cell signaling, 2775 and 3868), phospho-ULK1 (Cell signaling, 5869), phospho-AMPKα (Cell signaling, 2531), ULK1 (Cell signaling, 8054), AMPKα (Cell signaling, 5832), GFP (Santa Cruz, sc-9996), FLAG (Sigma, A8592), GAPDH (Cell signaling, 5174), β-Actin (Cell signaling, 3700), α-Tubulin (Sigma, T9026).

Che L., Yang X., Ge C., El-Amouri S.S., Wang Q.E., Pan D., Herzog T.J, & Du C. (2019). Loss of BRUCE reduces cellular energy level and induces autophagy by driving activation of the AMPK-ULK1 autophagic initiating axis. PLoS ONE, 14(5), e0216553.

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Metformin and Phenformin Regulate TGF-β Signaling

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Metformin and phenformin were purchased from Sigma-Aldrich (St. Louis, MO, USA); human TGF-β1 and antibodies against β-actin, p-AMPKα Thr172, AMPKα, p-ACC Ser79, ACC, Slug, p-Smad3 Ser423/425, and Smad2/3 were from Cell Signaling Technology (Beverly, MA, USA); ELISA kits for human and mouse TGF-β1, antibodies against E-cadherin, and vimentin were from Abcam (Cambridge, MA, USA); monoclonal antibody against LKB1 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); growth factor-reduced Matrigel and monoclonal antibody against β-catenin were from BD Biosciences (San Jose, CA, USA); Lipofectamine 2000, 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibodies were from Life Technologies (Grand Island, NE, USA); Chamber slides was from EMD Millipore (Billerica, MA, USA).

Li N.S., Zou J.R., Lin H., Ke R., He X.L., Xiao L., Huang D., Luo L., Lv N, & Luo Z. (2015). LKB1/AMPK inhibits TGF-β1 production and the TGF-β signaling pathway in breast cancer cells. Tumour Biology, 37(6), 8249-8258.

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Western Blot Analysis of Signaling Pathways

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Preparation of whole cell lysates and Western blots were performed as previously described⁵⁴. Phosphatase Inhibitor (LC laboratories) was added to inhibit phosphatase activity. Antibodies used in this study were: HSP90 (H-114, 1:6,000) and JNK1/2 (sc-571, 1:2,000) from Santa Cruz; p-Thr202/Tyr204 ERK1/2 (4370, 1:2,000), ERK1/2 (9102, 1:2,000), p-Ser217/221 MEK1/2 (9121, 1:1,000), MEK1/2 (4694, 1:1,000), p-Thr183/Tyr185 JNK1/2 (9255S, 1:2,000,), p-Ser473 AKT (9271S, 1:2,000), AKT (9272, 1:2,000), IκBα (9242, 1:2,000), p-Ser536 NFκB p65 (3033, 1:1,000), p-Thr172 AMPKα (2535, 1:2,000) and AMPKα (2532, 1:2,000) from Cell Signaling. Band density was quantitated using the Image Lab software on the ChemiDOC XRS+ system (Bio-Rad). Protein levels were normalized to HSP90 and the phosphorylated forms were normalized to phosphorylation-independent levels of the same protein. Data are presented as mean ± SEM unless otherwise specified. Uncropped blots are shown in Supplementary Fig. 9–11.

Ji Y., Sun S., Shrestha N., Darragh L.B., Shirakawa J., Xing Y., He Y., Carboneau B.A., Kim H., An D., Ma M., Oberhozler J., Soleimanpour S.A., Gannon M., Liu C., Naji A., Kulkarni R.N., Wang Y., Kersten S, & Qi L. (2019). Toll-like receptors TLR2 and TLR4 block the replication of pancreatic β cells in diet-induced obesity. Nature immunology, 20(6), 677-686.

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Western Blot Analysis of Cellular Proteins

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Cells were lysed in lysis buffer (20 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium pyrophosphate, Protease Inhibitor Cocktail) for 30 min on ice. The supernatant was collected after centrifugation at 14,000×g for 20 min at 4°C. Protein concentrations were determined using the BCA protein assay kit. Proteins were separated on the 12% SDS-PAGE gel and transferred onto a PVDF transfer membrane. Western blot analysis followed a standard procedure. NDUFA4, ENO2, MCT1 and β-catenin antibodies were obtained from Sigma (St Louis, MO). AMPKα, phospho-AMPKα (Thr172), PDHA1, E-cadherin, N-cadherin and β-actin were obtained from Cell Signaling Technology (Danvers, MA). The antibody for cytokeratin 18 was obtained from Millipore (Boston, MA). HIF1α antibody was obtained from Abcam (Cambridge, MA). Vimentin antibody was obtained from Proteintech (Chicago, IL). NDUFA5 and ADHFE1 antibodies were obtained from Pierce (Rockford, IL). HSP60 antibody was obtained from Stressgen (Victoria, BC).

Tang H., Chen Y., Liu X., Wang S., Lv Y., Wu D., Wang Q., Luo M, & Deng H. (2016). Downregulation of HSP60 disrupts mitochondrial proteostasis to promote tumorigenesis and progression in clear cell renal cell carcinoma. Oncotarget, 7(25), 38822-38834.

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Comprehensive Western Blot Protocol

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Antibodies from Cell Signaling Technology are as follows: AMPKα (1:1000; #2532), p-AMPK^Thr172 (1:1000; #2531), ATG13 (1:1000; #13468), Crkl (1:500; #3182), p-Crkl^Tyr207 (1:100; #3181), mTOR (1:500; #2983), p-mTOR^Ser2448 (1:500; #2971), RPS6 (1:1000; #2317), p-RPS6^Ser240/244 (1:1000; #5364), ULK1 (1:1000; #8054), p-ULK1^Ser757 (1:500; #6888), p-ULK1^Ser555 (1:500; #5869), p-ATG13S^er318/ATG^Ser355 (1:1000; #46329), LC3B (1:500; #2775), ATG7 (1:1000; #8558), glyceraldehyde phosphate dehydrogenase (GAPDH) (1:1000; #5174), β-tubulin (1:1000; #2146), mouse immunoglobulin G (IgG) (1:5000; #7076), and rabbit IgG (1:5000; #7074). OXPHOS cocktail (1:2000; Abcam, #110413), green fluorescent protein (1:1000; Roche, #118144600001), MT-CO2 (1:2000; Thermo Fisher Scientific, #A-6404), p62 (1:1000; BD, #610832), and p-ATG13^Ser318 (1:1000; Abnova, #NBP2-19127) were also used.

Ianniciello A., Zarou M.M., Rattigan K.M., Scott M., Dawson A., Dunn K., Brabcova Z., Kalkman E.R., Nixon C., Michie A.M., Copland M., Vetrie D., Ambler M., Saxty B, & Helgason G.V. (2021). ULK1 inhibition promotes oxidative stress–induced differentiation and sensitizes leukemic stem cells to targeted therapy. Science translational medicine, 13(613), eabd5016.

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Hippocampal Protein Extraction and Analysis

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Individual hippocampi were sonicated in lysis buffer (10mM Tris, pH7.4, 100 mM NaCl, 1 mM EDTA, 1mM EGTA, 1mM NaF, 20 mM Na 4 P 2 O 7, 2mM Na 3 VO 4, 1% Triton X-100, 10 % glycerol, 0.1% SDS, and 0.5% deoxycholate) with Complete protease inhibitor cocktail (Roche, Indianapolis, IN) as we have previously published (14 (link)). Reducing agent (Thermo Scientific, Waltham, MA) and LDS (Thermo) were added to each sample. 20–30 μg of total protein was separated using NuPAGE 4–12% Bis-Tris gels (Thermo) and transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA) as previously described (13 (link)). Using Rockland (Pottstown, PA) Blocking Buffer for Fluorescent Western Blotting and the following antibodies, the blots were imaged and analyzed with Odyssey infrared scanning (LiCor Bioscience, Lincoln, NE) as previously described. Mouse monoclonal anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO) and used at 1:10,000. Primary antibodies purchased from Cell Signaling Technology (Danvers, MA) and used at 1:500 included: pS6K (Thr389, #9205), S6K (#9202), pAkt (Ser473, #9271), pAkt (Thr308, #9275), Akt (#9272), p-mTOR (Ser2448, #2971), mTOR (#2972), p AMPKα (Thr172, #2531), AMPKα (#2532). Secondary antibodies were Alexa Fluor 680 conjugated anti-mouse IgG (1:12,500, Thermo) and Infrared Dye 800 conjugated anti-rabbit IgG (1:12,500, Rockland).

Wallin D.J., Zamora T.G., Alexander M., Ennis K.M., Tran P.V, & Georgieff M.K. (2017). Neonatal mouse hippocampus: Phlebotomy-induced anemia diminishes and treatment with erythropoietin partially rescues mammalian target of rapamycin (mTOR) signaling. Pediatric research, 82(3), 501-508.

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Comprehensive Antibody Panel for Cellular Analysis

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Antibodies to RKIP (CST#5291), AMPKα (CST#5832), phospho-AMPKα (T172) (CST#8208), p70S6K (CST#2708), phospho-p70S6K (CST#9208), 4EBP1 (CST#9644), phospho-4EBP1 (CST#9459), STAT3 (CST#9132), phospho-STAT3 (Tyr705) (CST#9131) and phospho-STAT3 (Ser727) (CST#9134) were purchased from Cell Signaling Technology. Antibodies to E-Cadherin (20874-1-AP) and vimentin (10366-1-AP) were obtained from Proteintech Group. Anti-ANXA7 antibody (sc-17815) used for co-immunoprecipitation and anti-p-Thr antibody (sc-5267) were obtained from Santa Cruz Biotechnology. Anti-ANXA7 antibody (A4475 MSDS) used for western blot and antibody to β-actin (A1978 MSDS) were purchased from Sigma. Horseradish peroxidase-conjugated secondary antibodies for Western blot were from Santa Cruze Biotechnology. Secondary antibody for immunofluorescence was donkey anti-rabbit IgG Alexa Fluor-488 (CA21206s, Invitrogen).

Liu S., Li X., Lin Z., Su L., Yan S., Zhao B, & Miao J. (2017). SEC-induced activation of ANXA7 GTPase suppresses prostate cancer metastasis. Cancer letters, 416, 11-23.

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