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Ampkα2

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AMPKα2 is a catalytic subunit of the AMP-activated protein kinase (AMPK) enzyme. AMPK is a cellular energy sensor that plays a key role in regulating metabolism. AMPKα2 is responsible for the kinase activity of the AMPK enzyme complex.

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Lab products found in correlation

Ampkα1, by Cell Signaling Technology (9 mentions) Ampkα, by Cell Signaling Technology (4 mentions) Nanodrop 2000, by Thermo Fisher Scientific (4 mentions) β actin, by Merck Group (3 mentions) Vinculin, by Merck Group (3 mentions) Phospho ampkα, by Cell Signaling Technology (3 mentions) Phospho acc, by Cell Signaling Technology (3 mentions) Ampkα1 2, by Cell Signaling Technology (2 mentions) Hif 1α, by Cell Signaling Technology (2 mentions) P acc, by Cell Signaling Technology (2 mentions) Vinculin, by Cell Signaling Technology (2 mentions) Phospho ampkα thr172, by Cell Signaling Technology (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Slc7a11, by Cell Signaling Technology (2 mentions) Phospho s6k, by Cell Signaling Technology (2 mentions) Phospho ulk1, by Cell Signaling Technology (2 mentions) Acsl4, by Santa Cruz Biotechnology (2 mentions) Phospho s6, by Cell Signaling Technology (2 mentions) Ripa lysis buffer, by Merck Group (2 mentions) Cleaved parp, by Cell Signaling Technology (2 mentions)

15 protocols using ampkα2

1

Western Blot Analysis of AMPK Signaling

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Cell Signaling Antibodies used at 1:1000 in 5% BSA in TBS-T: phospho-AMPKα1/2 Thr172 (#2535), AMPK α1/2 (#2532), AMPKα2 (#2757), phospho-ACC Ser79 (#3661), ACC (#3662), phospho-Raptor Ser792 (#2083), Raptor (#2280). Mouse monoclonal hAMPKα2 (#MAB2850) was purchased from R&D Systems. Flag M2 agarose resin (A2220), β-actin (A5441), and Flag M2 monoclonal (F1804) were purchased from Sigma. Phenformin hydrochloride was obtained from Sigma (P7045) and dissolved in SILAC DMEM (Thermo scientific) with 10% Dialyzed FBS (GIBCO-Life Technologies). Pierce High pH Reverse-Phase Peptide Fractionation Kit was purchased from Thermo Scientific. Acclaim PepMap 75 μm x 2 cm C18 trap columns (#164535) and Acclaim PepMap RSLC 75 μm x 50 cm analytical columns (#164942) were also purchased from Thermo Scientific
Stein B.D., Herzig S., Martínez-Bartolomé S., Lavallée-Adam M., Shaw R.J, & Yates JR I.I.I. (2019). Comparison of CRISPR Genomic Tagging For Affinity-Purification and Endogenous Immunoprecipitation coupled with Quantitative MS to Identify the Dynamic AMPKα2 Interactome. Journal of proteome research, 18(10), 3703-3714.
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2

Western Blot Analysis of Metabolic and Inflammatory Markers

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As described previously [34 (link)], equal amounts of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrophoretic transfer of proteins onto nitrocellulose membranes. Blots were probed with antibodies against AMPKα2 (Cell Signaling Technology), pAMPKα2 (Millipore, St. Louis, MO, USA), pACC (Millipore, St. Louis, MO, USA), PGC-1α (Abcam, Cambridge, MA, USA), CPT-1β (Abcam, Cambridge, MA, USA), CD36 (Santa Cruz Biotechnology, CA, USA), GLUT4 (Abcam, Cambridge, MA, USA), pIRS1 (Ser 307, Cell Signaling Technology), pAkt (Ser 473, Cell Signaling Technology), PI3K/Akt (Cell Signaling Technology), NLRP3 (Novus Biologicals), TNF-α (Santa Cruz Biotechnology, CA, USA), IL-1β (Cell Signaling Technology), pro-caspase-1 (Novus Biologicals), cleaved caspase-1 (Novus Biologicals), STAT3 (Cell Signaling Technology), pSTAT3 (Cell Signaling Technology), pERK1/2 (Cell Signaling Technology), and secondary antibodies conjugated with horseradish peroxidase (Leinco Technology, St. Louis, MO, USA). Bound antibodies were detected using an enhanced chemiluminescence detection system (Millipore, St. Louis, MO, USA) and analyzed with AlphaEaseFC software (Alpha Innotech, San Leandro, CA, USA). Targeted bands were normalized to cardiac glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Sigma-Aldrich) to confirm equal protein loading.
Trang N.N., Chung C.C., Lee T.W., Cheng W.L., Kao Y.H., Huang S.Y., Lee T.I, & Chen Y.J. (2021). Empagliflozin and Liraglutide Differentially Modulate Cardiac Metabolism in Diabetic Cardiomyopathy in Rats. International Journal of Molecular Sciences, 22(3), 1177.
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3

Antibody-based Protein Analysis Protocol

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Antibodies against HIF-1α, c-Myc, USP28, ubiquitin (Ub), AMPK-α1, AMPK-α2, AMPK, phospho-AMPK (Thr172), Caspase-3, GSK-3β, phospho-GSK-3β (Ser9), XIAP, Beclin-1, p-LKB (S428), ULK (S555) and LC3-I/II were purchased from Cell Signaling Technology. The rest of the antibodies, such as anti-Atg5, anti-phospho-c-Myc (Thr58), anti-phospho-c-Myc (Ser62), LKB and anti-ASS1 were respectively purchased from Abgent, Santa Cruz Biotech, Abcam, and kindly provided by Polaris Pharmaceuticals, Inc. The immunoblots were visualized by ChemiDoc MP System (Bio-Rad) and quantitation was performed by densitometer and Image J.
Li Y.Y., Wu C., Chen S.M., Shah S.S., Wangpaichitr M., Feun L.G., Kuo M.T., Suarez M., Prince J, & Savaraj N. (2016). BRAF inhibitor resistance enhances vulnerability to arginine deprivation in melanoma. Oncotarget, 7(14), 17665-17680.
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4

Western Blot Analysis of AMPK Signaling

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Cells were washed once with ice-cold Dulbecco's phosphate-buffered saline and lysed directly in 1× protein sample buffer (62.5 mM Tris-Cl, pH 6.8, 10% glycerol, 2% SDS, 1% β-mercaptoethanol, and trace amounts of bromophenol blue), boiled, and stored at −80°C. Proteins were separated by SDS–PAGE on a 4–15% gradient Tris-HCl polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Blots were probed with the following primary antibodies overnight in Tris-buffered saline solution, pH 7.5, containing 0.1% Tween-20, 5% protease-free bovine serum albumin (BSA), and 50 mM NaF (TBST): pAMPK, pACC, tAMPK, tACC, pAMPK substrate, AMPK α1, AMPK α2, BRSK1, BRSK2 (1:1000; Cell Signaling; Danvers MA), and actin (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were incubated for 2 h with the following secondary antibodies in 5% nonfat dry milk in TBST: anti-goat and anti-rabbit horseradish peroxidase (1:5000; EMD Millipore, Billerica, MA). The enhanced chemiluminescence signal was detected with the FluorChem Q imaging system (Protein Simple, Santa Clara, CA).
Sample V., Ramamurthy S., Gorshkov K., Ronnett G.V, & Zhang J. (2015). Polarized activities of AMPK and BRSK in primary hippocampal neurons. Molecular Biology of the Cell, 26(10), 1935-1946.
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5

Rabbit Antibody Western Blotting

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Rabbit monoclonal antibodies for western blotting against β-actin (#4970), vinculin (#4650), AMPKα (#2532), AMPKα1 (#2795), AMPKα2 (#2757), ACC1 (#3676), p70S6K (#9202), Raptor (#2280), Akt (#9272) and against phospho-AMPK [T172 (#2535) or S485 (#2537)], phospho-ACC (S79) (#3661), phospho-p70S6K (T389) (#9205), phospho-Raptor (S792) (#2083), and phospho-Akt (S473) (#9271) were obtained from Cell Signaling Technology (Frankfurt, Germany). Mouse monoclonal antibodies against the HSV-1 proteins gB (ab6506) and ICP4 (ab6514) were obtained from Abcam (Cambridge, United Kingdom).
Doshi H., Spengler K., Godbole A., Gee Y.S., Baell J., Oakhill J.S., Henke A, & Heller R. (2023). AMPK protects endothelial cells against HSV-1 replication via inhibition of mTORC1 and ACC1. Microbiology Spectrum, 11(5), e00417-23.
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6

Immunoblot Analysis of Protein Signaling Pathways

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Immunoblot analysis was performed as previously described [73 (link)]. Briefly, the cells were lysed in modified RIPA buffer containing 50 mM HEPES, 0.3% NP-40, 75 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, and EDTA-free protease (Thermo Fisher Scientific, cat. no. 88266) and phosphatase inhibitors (Thermo Fisher Scientific, cat. no. 88667) on ice. 35 ug of proteins were resolved using 4%–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, cat. no. 4561096) and transferred to PVDF membranes (Bio-Rad, cat. no. 1620177). The transferred blot was blocked with 5% BSA for 30 min in TBS containing 0.05% Tween-20 at room temperature for 30 min. Then the membranes were immunoblotted with primary antibody Egr1 (Santa Cruz, cat. no. sc-189; 1:500), phospho-AMPKα (Thr172) (Cell Signaling, cat. no. 2535; 1:1,000), AMPKα 1/2 (total) (Cell Signaling, cat. no. 2532; 1:1,000), AMPKα2 (total) (Cell Signaling, cat. no. 2757; 1:1,000), and β-tubulin (Cell Signaling, cat. no. 2146; 1:1,000) at 4°C overnight.
Di Biase S., Shim H.S., Kim K.H., Vinciguerra M., Rappa F., Wei M., Brandhorst S., Cappello F., Mirzaei H., Lee C, & Longo V.D. (2017). Fasting regulates EGR1 and protects from glucose- and dexamethasone-dependent sensitization to chemotherapy. PLoS Biology, 15(3), e2001951.
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7

Antibody-based Protein Detection Assays

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Western blots and immunohistochemical staining were performed as described.28, 29, 30 Antibodies used were SLC40A1 (NBP1‐21502, Novus Biologicals, Centennial, CO, USA, 1:500), GAPDH (sc‐32233, Santa Cruz Biotechnology, Dallas, TX, USA, 1:2000), AMPKα1 (2795, Cell Signaling Technology, Danvers, MA, USA, 1:1000 and sc‐19128, Santa Cruz Biotechnology, 1:500), AMPK‐α2 (2757, Cell Signaling Technology, 1:1000 and sc‐19131, Santa Cruz Biotechnology, 1:1000), pAMPK (Thr172, 2535, Cell Signaling Technology, 1:1000), Smad1/5/8 (sc‐6031, Santa Cruz Biotechnology, 1:1000), pSmad1/5/8 (9511, Cell Signaling Technology, 1:1000), HIF1α (14179, Cell Signaling Technology, 1:1000), Hydroxy‐HIF1α (Pro564) (3434, Cell Signaling Technology, 1:1000), pSer/Thr (ab17464, Abcam, 1:1000 ), PHD2 (4835, Cell Signaling Technology, 1:1000), HA (3724, Cell Signaling Technology, 1:1000) and His (12698, Cell Signaling Technology, 1:1000).
Wang C., Zhang W., Xu W., Liu Z, & Huang K. (2022). AMP‐activated protein kinase α1 phosphorylates PHD2 to maintain systemic iron homeostasis. Clinical and Translational Medicine, 12(5), e854.
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8

Antibodies for Protein Phosphorylation Analysis

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The antibodies used were directed against p-Ser1177 (Cell signaling, Cat. No. 9571) and p-Thr495 eNOS (Cell signaling, Cat. No. 9574), eNOS (BD Transduction, 610296), p-Thr172 AMPK (Cell signaling, Cat. No. 2535), AMPKα2 (Cell signaling, Cat. No. 2757), β-actin (Sigma, Cat. No. A5441), Flag (Sigma, Cat. No. F3165), and c-Myc (Santa Cruz, Cat. No. SC-40). The AMPKα1 antibody was generated by Eurogentec by injecting rabbits with the AMPKα1-specific peptide H2N–CRA RHT LDE LNPQKS KHQ–CONH2. All other substances were obtained from Sigma-Aldrich (Munich, Germany). 32Pγ-ATP was obtained from Hartmann Analytics (Braunschweig, Germany).
Zippel N., Loot A.E., Stingl H., Randriamboavonjy V., Fleming I, & Fisslthaler B. (2018). Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation. International Journal of Molecular Sciences, 19(9), 2753.
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9

Quantitative Western Blot Analysis of Protein Expression

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Western blotting to analyze protein expression was performed as previously described62 –64 (link). Briefly, cell pellets and tissues were lysed using RIPA lysis buffer (Millipore) and the protein concentration was determined by a Bradford assay (Bio-Rad) using a Nanodrop2000 (Thermo scientific). 15–20 μg of protein was used for immunoblot analysis using antibodies against phospho-AMPKα (Thr172, 1:1000, 2535, Cell Signaling), AMPKα (1:1000, 5832, Cell Signaling) AMPK α1 (1:1000, 2795, Cell Signaling), AMPKα2 (1:1000, 2757, Cell Signaling), phospho-ACC (S79, 3661, 1:1000, Cell Signaling), ACC (1:1000, 3662, Cell Signaling), phospho-S6 (Ser240/244, 1:5000, 3661, Cell Signaling), S6 (1:1000, 2217, Cell Signaling), phospho-S6K (Thr389, 1:1000, 9205, Cell Signaling), S6K (1:1000, sc-230, Santa Cruz), phospho-ULK1 (S757, 6888, 1:1000, Cell Signaling), ULK1 (1:1000, 8054, Cell Signaling), LKB1 (1:1000, sc-32245, Santa Cruz), SLC7A11 (1:3000, 12691, Cell Signaling), GPX4 (1:1000, MAB5457, R&D systems), ACSL4 (1:1000, sc-271800, Santa Cruz), Cleaved Caspase-3 (Asp175, 1:500, 9661, Cell Signaling), Cleaved-PARP (Asp214, 1:1000, 9544, Cell Signaling), Vinculin (1:50000, V4505, Sigma).
Lee H., Zandkarimi F., Zhang Y., Meena J.K., Kim J., Zhuang L., Tyagi S., Ma L., Westbrook T.F., Steinberg G.R., Nakada D., Stockwell B.R, & Gan B. (2020). Energy stress-mediated AMPK activation inhibits ferroptosis. Nature cell biology, 22(2), 225-234.
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10

Comprehensive Immunoblotting Antibody Protocol

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Phospho-AMPKα (1:1000, Thr 172) (2535), AMPKα (1:1000, 2603), AMPKα1 (1:1000, 2795), AMPKα2 (1:1000, 2757), AMPKβ1 (1:1000, 4178), AMPKγ1 (1:1000, 4187), phospho-ACC (1:1000, 3661), ACC (1:1000, 3676), phospho-AKT (1:1000, Ser 473) (9271) and AKT1 (1:2000, 2967) antibodies were acquired from Cell Signaling Technology (Danvers, MA, USA). The UCP1 antibody (1:1000, ab10983) and mouse AMPKα1 (1:1000, ab3759) were procured from Abcam (Cambridge, MA, USA). Mouse AMPKα2 (1:1000, AF2850) was purchased from R&D systems Inc. (MN, USA). FLAG (1:3000, F3165 and F7425) and β-actin (1:10000, A5316) antibodies were procured from Sigma-Aldrich. The MKRN1 antibody (1:3000, A300-990A) was procured from Bethyl Laboratories. The mono- and polyubiquitin chain antibodies (1:1000, FK2, Biomol, PW0150) were purchased from Enzo Life Sciences. The HA antibody (1:3000, 12013819001) was obtained from Roche. GAPDH (1:5000, sc-25778), GFP (1:5000, sc-8334) and HA (hemagglutinin) (1:3000, sc-7392 and sc-805) antibodies were procured from Santa Cruz Biotechnology (Dallas, TX, USA). MG132 (M-1157) was purchased from A.G. Scientific (CA, USA). CHX (C4859) was purchased from Sigma-Aldrich.
Lee M.S., Han H.J., Han S.Y., Kim I.Y., Chae S., Lee C.S., Kim S.E., Yoon S.G., Park J.W., Kim J.H., Shin S., Jeong M., Ko A., Lee H.Y., Oh K.J., Lee Y.H., Bae K.H., Koo S.H., Kim J.W., Seong J.K., Hwang D, & Song J. (2018). Loss of the E3 ubiquitin ligase MKRN1 represses diet-induced metabolic syndrome through AMPK activation. Nature Communications, 9, 3404.
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