Cells treated or not with metformin (10mM), were lysed in 70μL of lysis buffer (50mM Tris HCl pH 7.5, 0.1% Triton, 5mM EDTA complemented by protease (Chemicon Millipore) and phosphatase (Sigma-Aldrich) inhibitors. Western blots were performed as previously described using monoclonal rabbit antibodies [20 (link), 22 (link)], anti-LC3b (1/1000, Cell Signaling), anti-Beclin 1 (1/1000, Cell Signaling), anti-p62 (1/1000 Abcam), anti-phospho (T172) AMPK (1/1000, Cell Signaling), anti-AMPK (1/1000, Cell Signaling) anti-phospho (S79) ACC (1/1000, Cell Signaling), anti-ACC (1/1000, Cell Signaling), anti-phospho (S2448) mTOR (1/1000, Cell Signaling), anti-mTOR (1/1000, Cell Signaling), anti-phospho (T389) p70S6 Kinase (1/1000, Cell Signaling), anti-p70S6 Kinase (1/1000, Cell Signaling), anti-phospho (T37/46) 4EBP1 (1/1000, Cell Signaling), anti-phospho (S473) AKT (1/1000, Cell Signaling), anti-phospho (T308) AKT (1/1000, Cell Signaling), anti-AKT (1/1000, Cell Signaling), anti-HIF-1α (1/1000, Cayman Chemical), anti-Redd1/DDIT4 (1/1000, Abcam and Proteintech) and were normalized using a rabbit polyclonal antibody anti-β-tubulin (1/1000, Cell Signaling). Gel quantification was performed using ImageJ (Windows 1.47, Research Services Branch, NIH).
Anti phospho ampk t172
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-AMPK T172 is a primary antibody that recognizes the threonine 172 (T172) phosphorylated form of the AMP-activated protein kinase (AMPK) enzyme. AMPK is a sensor of cellular energy status and plays a crucial role in regulating metabolism and energy homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Anti acc, by Cell Signaling Technology (4 mentions)
Anti ampk, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti tubulin, by Cell Signaling Technology (2 mentions)
Anti rabbit immunoglobulin g igg horseradish peroxidase hrp linked antibody, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti mcherry, by Abcam (2 mentions)
Anti atg5, by Cell Signaling Technology (2 mentions)
Anti phospho acc s79, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti hif 1α, by Cayman Chemical (1 mentions)
Anti p70 s6 kinase, by Cell Signaling Technology (1 mentions)
Anti beclin 1, by Cell Signaling Technology (1 mentions)
Anti phospho akt s473, by Cell Signaling Technology (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Anti phospho akt t308, by Cell Signaling Technology (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Anti p62, by Abcam (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti phospho ampk t172
1
Metformin Effects on Autophagy Signaling
2
Quantitative Western Blot Analysis of Muscle Proteins
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Muscles were homogenized by hand in a Dounce homogenizer in reducing sample buffer (80 mM Tris, pH 6.8, 10% β-Mercaptoethanol, 2% SDS and 10% glycerol) with phosphatase inhibitor (Thermo scientific) and protease inhibitor cocktail (Sigma). An equal amount of total protein was loaded on SDS-PAGE gels followed by transfer to nitrocellulose membrane. Ponceau red S was used to verify the transfer onto the nitrocellulose. The following primary antibodies were used for western blotting: anti- β-CaMKII (1:500, Life Technologies), anti- phospho-CaMKII (1:750, Thermo Scientific), anti-AMPK (1:1000), anti-phospho (T172) AMPK (1:1000), anti-Akt (1:1000), anti-phospho Akt (1:500)(Cell Signaling). Secondary antibodies conjugated with HRP were from Sigma-Aldrich (used 1:10000). Specific signals were developed using ChemiGlow chemiluminescent substrate for HRP (Protein Simple). Images of the blots were acquired using FluorChem FC2 Imager (Alpha Innotech). Specific signals were developed using ChemiGlow chemiluminescent substrate for HRP (Protein Simple). Images of the blots were acquired using FluorChem FC2 Imager (Alpha Innotech). Quantitative analysis was performed using ImageJ software.
3
Regulation of Endothelial Nitric Oxide Synthase
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Fura-2 AM was purchased from Teflabs (Austin, TX, USA) and Cal-520 AM from Stratech (Newmarket, United Kingdom). eNOS, PP2A-A (α/β), and PR72/130 antibodies were from Santa Cruz Biotechnologies (Dallas, TX, USA). Anti-PP2A-C and α-tubulin were from Millipore-Sigma (Watford, United Kingdom). Anti-phospho-eNOS S1177, T495, and S633; anti-phospho-AMPK T172; and total α-AMPK, anti-phospho-protein kinase B (Akt) S473 and total Akt, anti-phospho-ERK1/2 (T204/202) and total ERK1/2 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-HIF1-α antibody was from Novus Biologicals (Littleton, CO, USA). cGMP ELISA was from Cayman Chemical (Ann Arbor, MI, USA). The anti-SERCA-2 antibody (2D8) was kindly provided by Dr. Kalwant Authi (Cardiovascular Division, King’s College London). On-Target Plus PP2A-C (human) and control scrambled siRNA were from Dharmacon (Lafayette, CO, USA). All other chemicals were purchased from Millipore-Sigma.
4
Western Blot Analysis of AMPK Activation
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Cells were homogenized in a solution of 50 mM Tris-HCl pH 7.5, 125 mM NaCl, 1% Nonidet P40, 5 mM NaF, 1.4 mM Na4O7P2, 1 mM Na3VO4, and protease inhibitor (Fisher Scientific, Madrid, Spain). The homogenate was then centrifuged at 600× g for 5 min at 4 °C. Protein determination was performed using the bicinchoninic acid protein assay (Pierce Biotechnology, Waltham, MA, USA). Whole cell lysates (20–30 μg) were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Barcelona, Spain). After blocking, the blots were incubated with the following antibodies: anti-phospho-AMPK (T172) (Cell Signaling Technology, Danvers, MA, USA), anti-AMPK (Cell Signaling Technology), and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by a secondary antibody conjugated to IRDye 800 CW or IRDye 680 LT (LI-COR, Lincoln, NE, USA). The membranes were then analyzed using the Odyssey Infrared Imaging System (LI-COR). Densitometric analysis was performed using the Odyssey Infrared Imaging System, version 3.0 (LI-COR).
5
Western Blotting Analysis of Cellular Proteins
6
Antibody-Based Protein Analysis Protocol
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Anti-Sirt1 (#2493, 1:1000), anti-LKB1 (#13031, 1:1000), anti-acetyl-p53 K382 (#2525, 1:1000), anti-p53 (#18032, 1:1000), anti-phospho-AMPK T172 (#2535, 1:1000), anti-AMPK (#5831, 1:1000), anti-phospho-ACC Ser79 (#11818, 1:1000), anti-ACC (#3662, 1:1000), anti-MO25α (#2716, 1:2000), anti-acetylated-lysine (#9441, 1:1000), anti-Lamin B1 (#13435, 1:1000), anti-GST-tag (#2625, 1:2000), anti-His-tag (#12698, 1:2000), anti-HA-tag (#3724, 1:2000), and anti-MBP-tag (#2396, 1:2000) antibodies were from Cell Signaling Technology. Anti-phospho-Ser/Thr (ab17464, 1:1000) and anti-α-tubulin (ab80779, 1:5000) antibodies were from Abcam. Anti-STRAD (N-13) (sc-34102) was from Santa Cruz. Anti-FLAG-tag (M2, 1:2000) antibody was from Sigma. Lambda Protein Phosphatase (P0753) was from New England Biolabs. Resveratrol, piceatannol, quercetin, fisetin, EX527, nicotinamide, NAD, N-ε-acetyl-L-lysine, SRT1720, 3xFLAG peptide, doxorubicin, phosphatase inhibitors, and protease inhibitors were from Sigma.
7
Western Blotting Analysis of Cellular Proteins
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Cells were lysed in ice-cold RIPA buffer and western blotting was performed as described previously (Florey et al., 2011 (link)). The following antibodies were used: anti-E-cadherin (1:500; 3195, Cell Signal), anti-tubulin (1:2,000; 3873, Cell Signal), anti-Atg5 (1:500; 2630, Cell Signal), anti-phospho-ACC-S79 (1:500; 3661, Cell Signal), anti-ACC (1:500; 3662, Cell Signal), anti-phospho-AMPK-T172 (1:500; 2531, Cell Signaling), anti-mCherry (1:500; ab125096, Abcam), anti-β-actin (1:2000; A1978, Sigma-Aldrich), anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody (1:5,000; 7074, Cell Signal), and anti-mouse IgG HRP-linked antibody (1:5,000; 7076, Cell Signal).
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