Protein lysates were separated on polyacrylamide gels followed by transfer onto nitrocellulose membranes. Membranes were incubated with antibodies against phospho-ACC (Ser79) (#3661), phospho-AMPK (Thr172) (#2531), phospho-eIF2α (Ser51) (#3398), phospho-cJun (Ser73) (#3270), phospho-S6 ribosomal protein (Ser240/244) (#2215), ACC (#3662), AMPK (#2532) (Cell Signalling Technology), sXBP1 (6196, BioLegend), phospho-IRE1 (Ser724) (ab124945, Abcam), ATF6 (NBP1-75478, Novus Biologicals), ATF4 (sc-200), or nucleolin (sc-13057) (Santa Cruz Biotechnology) followed by IRDye 800-coupled secondary antibodies (LICOR Biosciences). Blots were visualized and quantified using the Odyssey imaging system (LICOR Biosciences).
Phospho ampk thr172
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Phospho-AMPK (Thr172) is a laboratory product that detects the phosphorylation of AMP-activated protein kinase (AMPK) at threonine 172. AMPK is a key sensor of cellular energy status and plays a crucial role in regulating metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Total ampk, by Cell Signaling Technology (9 mentions)
Phospho acc ser79, by Cell Signaling Technology (7 mentions)
β actin, by Merck Group (6 mentions)
Phospho mtor ser2448, by Cell Signaling Technology (5 mentions)
Phospho akt ser473, by Cell Signaling Technology (5 mentions)
Phospho eif2α ser51, by Cell Signaling Technology (3 mentions)
Rapamycin, by Merck Group (3 mentions)
Phospho p70s6k thr389, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Phospho akt thr308, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
β actin, by Abcam (3 mentions)
Pgc 1α, by Novus Biologicals (3 mentions)
Compound c, by Merck Group (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Phospho ulk1 ser555, by Cell Signaling Technology (2 mentions)
53 protocols using phospho ampk thr172
1
Immunoblotting Quantification of Cellular Signaling
2
Whole-Heart Protein Lysate Analyses
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Whole‐heart protein lysates made from freeze‐clamped hearts using lysis buffer (HEPES pH 7.4 [20 mmol/L], β‐glycerol phosphate [50 mmol/L], EGTA [2 mmol/L], DTT [1 mmol/L], NaF [10 mmol/L], NaVO4 [1 mmol/L], Triton X‐100 [1%], glycerol [10%,] and 1 protease inhibitor complete mini tablet‐EDTA free/20 mL [Roche]) were subjected to SDS‐PAGE with protein content quantified using the Bradford method (Bio‐Rad). Following transfer, nitrocellulose membranes were probed with primary antibodies (LKB1 [D60C5; Cell Signaling]), LKB1 (M‐18; Santa Cruz Biotechnology), phospho‐LKB1 (Ser428/431; Cell Signaling), phospho‐AMPK (Thr172; Cell Signaling), total AMPK α2 (Santa Cruz Biotechnology), GAPDH (Abcam), STRAD (S‐17; Santa Cruz Biotechnology), MO25 (Cell Signaling), VDAC (Abcam), PUMA (Cell Signaling), SNRK (Sigma‐Aldrich), cleaved and total caspase 3 (Cell Signaling), and visualized using LI‐COR secondary antibodies and the LI‐COR Odyssey IR imager. Densitometry was quantified using Odyssey software. Primary antibody directed against p53 (Cell Signaling) was detected using enhanced chemiluminescence and quantified using MultiGauge software (Fujifilm).
3
Western Blot Analysis of Protein Markers
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Protein was extracted from cultured cells using cell scraper and RIPA buffer with protease and phosphatase inhibitor. Approximate amounts of protein were subjected to SDS‐PAGE and transferred to membranes. The membranes were incubated with the primary antibodies, including actin (1:6000, MAB1501, Millipore), Drp1 (1:1000, ab56788, Abcam), phospho‐Drp1 (S616) (1:1000, 3455, Cell Signaling), AMPK (1:1000, 2532, Cell Signaling), phospho‐AMPK (Thr172) (1:500, 2535, Cell Signaling), Bax (1:1000, ab32503, Abcam) and PARP (1:1000, 9542, Cell Signaling). Horseradish peroxidase (HPR)‐conjugated secondary antibody was used for binding the primary antibody. Binding bands were visualized by enhanced chemiluminescence (WBLUF0500, Millipore) and quantified using ImageJ software.
4
Quercetin Modulates Macrophage Metabolism
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Quercetin and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). The mouse macrophage-like cell line, RAW264.7 (CSTR:19375.09.3101MOUTCM13), was acquired from the National Collection of Authenticated Cell Cultures, the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s modified eagle’s medium (DMEM) high glucose were purchased from Gibcol Life Technology (Thermo Fisher, Waltham, MA, USA). An enhanced ATP Assay Kit was acquired from Beyotime® Biotechnology (Shanghai, China). anti-PGC1 (Cat# ab191838), anti-glutathione peroxidase 4 (Cat# ab125066), anti-AMPK alpha 1 (Cat# ab32047) and anti-SIRT1 (Cat# ab110304) antibodies were purchased from Abcam Biotechnology (Cambridge, MA, USA). Phospho-AMPK (Thr172) (Cat# 2535s) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The 3-4,5-dimethylthiazole-z-yl-3,5-diphenyltetrazolium bromide (MTT), trypsin, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), LA Assay Kit, Micro Pyruvate (PA) Assay Kit and Mitochondrial Membrane Potential Assay Kit with JC-1 were purchased from Solarbio Science & Technology Co. Ltd. (Beijing, China), and stored at −20 °C. EX-527 and compound C were obtained from Med Chem Express (Monmouth Junction, NJ, USA).
5
Quantifying Akt and AMPK Phosphorylation
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Isolated soleus muscles from 28 weeks old mice were used to determine the levels of Akt and AMPK phosphorylation. Muscles were homogenized in lysis buffer (10% Glycerol, 5% β-mercaptoethanol, 2.3% SDS, 62.5 mM Tris-HCl pH 6.8 and 6 M urea, supplemented with 1% phosphatase inhibitor cocktails 2 and 3 Sigma Aldrich) at a concentration of 10 mg of muscle/ml buffer. Subsequently, 50 µg of protein were separated on a 10% SDS PAG, transferred onto nitrocellulose (Amersham), and probed with the following primary antibodies: phospho-AMPK (Thr172), AMPK alpha, phospho-Akt (ser 473), phopho-Akt (thr 308) and Akt total from Cell Signaling and Desmin (used as housekeeping loading control) from Santa Cruz. The intensity of the immunopositive bands was determined using Image J.
6
Autophagic Cell Death Regulation
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Monodansylcadaverine (MDC); 3-methyladenine (3-MA); rapamycin; N-acetyl-l -cysteine (NAC); Compound C; 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA); and cisplatin were obtained from Sigma-Aldrich (St. Louis, MO, USA). LC3B and Atg5 antibodies were also purchased from Sigma-Aldrich. Control siRNA and Atg5-siRNA were obtained from Life Technologies (Carlsbad, CA, USA). Cl-caspase 3; mammalian target of rapamycin (mTOR); p-mTOR (Ser2448); AMPK; phospho-AMPK (Thr172); p70S6kinase (S6); and phosphor-S6 (Thr389) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SQSTM1/p62 and cl-PARP were obtained from Epitomics (Burlingame, CA, USA); and β-actin and CCK-8 were purchased from Beyotime Biotechnology (Shanghai, China). The FITC Annexin–V Apoptosis Detection Kit I was purchased from BD Biosciences (San Jose, CA, USA).
7
Protein Extraction and Western Blotting
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For protein extraction, tissue was homogenized in RIPA buffer containing protease and phosphatase inhibitor cocktail (#A32959, Thermo Scientific) and protein concentration was measured using the DC™ Protein Assay Reagent (#500‐0114, BioRad). The following primary antibodies were used: phospho‐AMPK (Thr172) (#2531, Cell Signaling Technology), total AMPK (#2603, Cell Signaling Technology), phospho‐eIF2α (Ser51) (#3597, Cell Signaling Technology), eIF2α antibodies (#3524, Cell Signaling Technology), OXPHOS antibody (#ab110413, Abcam), slow myosin (#ab11083, Abcam), and UCP1 antibodies (#ab23841, Abcam). Protein expression was normalized to α‐Tubulin (ATUB) (#T6074, SIGMA). Horseradish peroxidase‐conjugated secondary antibodies were used: anti‐rabbit IgG (#7074, Cell Signaling Technology) or anti‐mouse IgG (#7076, Cell Signaling Technology).
8
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.
9
Mesoglycan Modulates AMPK and mTOR Signaling
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Mesoglycan was kindly provided from Chodang Pham. CO.. Dulbecco’s modified eagle medium (DMEM) and fetal bovin serum (FBS) were purchased from Thermo Scientific (Logan, UT, U.S.A.). Pro-prep protein extract buffer was purchased from Intron Biotechnology (Sungnam, Korea). Antibodies against LKB1, phospho-LKB1 (Ser428), AMPK, phospho-AMPK (Thr172), phospho-ACC (Ser79), mTOR, phospho-mTOR (Ser2448), Bcl-2, Bax, phospho-p70S6K (Thr389) and phospho-4EBP1 (Ser65) were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). A monoclonal antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, MO). Compound C, AMPK inhibitor, was provided by Calbiochem (La Jolla, CA, U.S.A.). Human Platelet-Derived Growth Factor BB was purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). AMPK siRNA, Raptor siRNA, Control siRNA, p53, p27, p21, PCNA and Cytochrome C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).
10
Metformin Effects on Cellular Signaling Pathways
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Roswell Park Memorial Institute (RPMI) 1640 medium (2.0 g/L, 10 mM glucose), Low fasting glucose (FG) (1.0 g/L, 5 mM) or high glucose (HG) (4.5 g/L, 25 mM) Dulbeccos's modified eagle medium (DMEM), fetal bovine serum (FBS), trypsin/ethylenediaminetetraacetic acid (EDTA), and penicillin/ streptomycin were obtained from Gibco (Grand Island, NY, USA). Metformin was obtained from Cayman (Ann Arbor, MI, USA), and used following dissolution in phosphate buffered solution (PBS). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemicals (St. Louis, MO, USA). Bax, Bcl-2, TSC-2, mTOR, phospho-AMPKThr172, phospho-AMPKSer485, AMPK, phospho-Akt, Akt, and β-actin antibodies were obtained from Cell Signaling (Danvers, MA, USA). The Pro-IGF-1R and IRS-1 antibodies and goat anti-rabbit IgG-horseradish peroxidase (HRP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Luminate Forte Western HRP western Blotting Detection kit was obtained from Millipore (Danvers, MA, USA).
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