Homogenized skeletal muscle (0.1 g) at 4˚C in 1 ml of lysis buffer containing 50 mM Tris.HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 5 mM Na3VO4, 20 mM NaF, 10 mM sodium pyrophosphate and 50 µl protease inhibitor cocktail (B14001, Bimake). Equal quantities (50 µg) of total protein were resolved on a 12% gel using SDS-PAGE and transferred to a PVDF membrane (FFP32, Beyotime). After transfer, membranes were blocked using 5% skimmed milk containing 0.1% Tween-20. Subsequently the membranes were incubated with anti-GLUT-4 (2213S, Cell Signaling Technology, America), anti-phospho (p)-AMPK (2535S, Cell Signaling Technology), total-AMPK (2532S, Cell Signaling Technology) or anti-β-actin (ab8227, Abcam). Membranes were then incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies, including HRP-conjugated goat anti-rabbit immunoglobulin G (IgG) (AP132P, Merck Millipore), or HRP-conjugated goat anti-mouse IgG (ab6789, Abcam). Enhanced chemiluminescence plus kit (PE0010, Solarbio) was used to visualize the signals. Relative protein expression levels were quantified using densitometry analysis and normalized to β-actin expression levels.
Anti phospho ampk
Manufactured by Cell Signaling Technology
Sourced in United States, China
Anti-phospho-AMPK is a primary antibody that detects the phosphorylated form of the AMP-activated protein kinase (AMPK) enzyme. AMPK is a crucial cellular energy sensor that is activated in response to an increased AMP/ATP ratio, which can occur during cellular stress or nutrient deprivation. The phosphorylation of AMPK is a key indicator of AMPK activation.
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Lab products found in correlation
Anti ampk, by Cell Signaling Technology (50 mentions)
Anti phospho akt, by Cell Signaling Technology (16 mentions)
Anti akt, by Cell Signaling Technology (14 mentions)
Anti phospho mtor, by Cell Signaling Technology (11 mentions)
Anti β actin, by Santa Cruz Biotechnology (10 mentions)
Anti mtor, by Cell Signaling Technology (9 mentions)
Anti phospho acc, by Cell Signaling Technology (8 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (7 mentions)
Anti acc, by Cell Signaling Technology (6 mentions)
Anti 4e bp1, by Cell Signaling Technology (6 mentions)
Anti phospho 4ebp1, by Cell Signaling Technology (6 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (6 mentions)
Anti p70s6k, by Cell Signaling Technology (6 mentions)
Anti p38, by Cell Signaling Technology (5 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Anti gapdh, by Santa Cruz Biotechnology (5 mentions)
Anti erk1 2, by Cell Signaling Technology (5 mentions)
Anti β actin, by Merck Group (5 mentions)
Anti tubulin, by Merck Group (4 mentions)
Anti phospho p53, by Cell Signaling Technology (4 mentions)
67 protocols using anti phospho ampk
1
GLUT-4 and AMPK Expression in Skeletal Muscle
2
Kidney Protein Analysis by Western Blotting
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Protein from the kidney tissue and cultured cells was analysed using PRO‐PREP protein extraction solution (iNtRON Biotechnology). The concentration of the extracted protein was measured using a Bicinchoninic Acid Protein Assay Kit (Pierce, Rockford). Western blotting was performed on 8%–12% SDS‐PAGE. The blots were incubated with the following primary antibodies: anti‐arginase 2, anti‐cluster of differentiation 68 (CD68), anti‐nuclear factor, erythroid 2 like 2 (Nrf2), anti‐haeme oxygenase‐1 (HO‐1), anti‐NADPH oxidase (NOX) 4, sterol regulatory element‐binding proteins (SREBP) 1, SREBP2, acetyl‐CoA carboxylase (ACC), Bcl2, Bcl‐2‐associated X protein (BAX) and anti‐β‐actin (all from Santa Cruz Biotechnology) as well as anti‐nephrin (Abcam), anti‐phospho (p)‐AMPK, and anti‐p‐p38 and anti‐p38 (Cell Signaling, Danvers). The blots were visualized using a chemiluminescence UVP BioSpectrum 600 imaging system and quantified with ImageJ program (National Institute of Mental Health, Bethesda).
3
Quantification of AMPK and ACC Phosphorylation
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The phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase (ACC) were determined using Western blot. The protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk for 1 h at 37 C and incubated with the appropriate primary antibodies including anti-phospho-(p)-AMPK, anti-AMPK, anti-phospho-(p)-ACC, anti-ACC, anti-gp91 phox and GAPDH (Cell Signaling Technology) overnight at 4 C. After three washings with phosphate buffer solution containing 0.1% tween-20 (PBST), the membranes were subjected to corresponding secondary horseradish peroxidase-conjugated antibody incubation for 1 h at 37 C. After additional washings as described above, the blots were evaluated by densitometric analysis using an enhanced chemiluminescent system.
4
Western Blot Analysis of Cell Signaling
5
Cell Proliferation and Apoptosis Assay
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A cell counting kit (CCK) for WST assay was purchased from Donginbiotech Co. (Seoul, Korea). Crystal violet solution was purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-phospho-AMPK, phospho-p38, phospho-ERK, phospho-JNK, cyclin D1, CDK4, AMPK, PARP, caspase-3, Bcl-2, Bcl-xL, and Bax antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-p38, ERK, JNK, GAPDH, and α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
6
Synaptosome Fractionation and AMPK Analysis
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Tissues obtained from mice were pooled (two-three mice) and homogenized. The procedures for synaptosome fractionation and western blot analysis have been described in a previous study published by our group23 (link). The membranes were washed in the buffer and incubated with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG, 1:3000) for 2 h at 26 ℃. The optical density of each band was measured using Image J. Anti-AMPK (1:2000) and anti-phospho-AMPK (1:1000) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).
7
Quantitative Immunoblot Analysis of mTOR Signaling
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Equal amounts of protein were analyzed in duplicate by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The following monoclonal antibodies were used: anti–phospho-Akt (Ser473, Cell signaling, Danvers, MA), anti–total Akt (Cell signaling), anti-phospho-mTOR (Ser2448, Cell signaling), anti-phospho-mTOR (Thr2446, Millipore, Bedford, MA), anti-total mTOR (Cell signaling), anti-HIF-1α (Cayman Chemical, Ann Arbor, MI), anti-VHL (Cell signaling), anti-phospho-AMPK (Thr172, Cell signaling), anti-total AMPK (Cell signaling) anti-phospho-P70S6K (Thr389, Cell signaling), anti-total P70S6K (Cell signaling), anti-phospho-4E-BP (Thr37/46, Cell signaling), anti-lactate dehydrogenase A (LDHA) (Cell signaling), anti-hexokinase-2 (HK-2, SantaCruz Biotechnology, Dallas, TX), anti-phospho-TSC2 (Ser1387) (Cell signaling), anti-total TSC2 (Cell signaling), and anti-β-actin (Bethyl, Montgomery, TX, US). Immunoreactive proteins were detected by horseradish peroxidase–conjugated secondary antibodies and enhanced using chemiluminescence (ECL) reagents (GE Healthcare Life Sciences, Piscataway, NJ). All immunoblots were performed with triplicate and visualized by LAS image analyzer (Fujifilm, Tokyo, Japan). The band density was quantified using MultiGauge (Fujifilm).
8
Metformin and TRAIL-Induced Apoptosis
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Metformin was purchased from Wako (Richmond, VA, USA). TRAIL (Recombinant human) was purchased from Millipore (Millipore, Darmstadt, Germany.) Protein G PLUS-Agarose, Anti-Bax, anti-Bcl-2, anti-Mcl-1(IP), anti-Ub and anti-Bcl-xL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-phospho ERK, anti-ERK, anti-phospho JAK2, anti-JAK2, anti-phospho AMPK, anti-AMPK, anti-phospho mTOR, anti-mTOR, anti-phospho AKT, anti-AKT, anti-phsopho GSK3β, anti-GSK3β, anti-Noxa, anti-Puma, anti-Bim, anti-phospho Mcl-1, anti-Mcl-1(WB), anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-phospho STAT3, anti-STAT3, and anti-PARP-1 were purchased from Cell Signaling (Beverly, MA, USA). Anti-actin antibody was purchased from Sigma (Sigma, St. Louis, MO). Anti-Mule antibody was purchased from Abcam (Cat. No. ab70161). For the secondary antibodies, anti-mouse-IgG-HRP and anti-rabbit-IgG-HRP were purchased from Cell Signaling (Beverly, MA, USA).
9
Immunoblotting Analysis of Signaling Proteins
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We used the following antibodies: anti-Akt (No. 9272), anti-phospho-Akt (Ser473; No. 9271), anti-S6K (No. 9202), anti-phospho-S6K (Thr389; No. 9205), anti-S6 (No. 2217), anti-phospho-S6 (Ser235/236; No. 4858), anti-4EBP1 (No. 9644), anti-phospho-4EBP1 (Thr37/46; No. 2855), anti-GSK3β (No. 12456), anti-phospho-GSK3β (Ser9; No. 5558), anti-Ampk (No. 2532), anti-phospho-Ampk (Ser9; No. 2535), anti-mouse IgG (No. 7076), and anti-rabbit IgG (No. 7074) purchased from Cell Signaling Technology (Massachusetts, United States of America). Anti-α-Tubulin (12G10) was purchased from DSHB (Iowa, United States of America). Anti- Laminin α-2 (SC-59854) was purchased from Santa Cruz Biotechnology (Texas, United States of America).
10
Western Blot Antibody Reagents for Signaling Pathways
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Anti-AMPK (#2793) and anti-phospho-AMPK (#2535) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies for detecting phosphatidylinositol-3-kinase (PI3K; #06–497), AKT (#07–416), phospho-AKT (#07–310), p53 (#CBL404), matrix metalloproteinase-2 (MMP-2; #AB19015), and MMP-9 (#AB19016) were from Millipore (Bedford, MA, USA). The antibody for recognizing GAPDH (#NB300-221) was obtained from Novus Biologicals (Littleton, CO, USA). Anti-rabbit (#GTX213110-01) and anti-mouse (#ab6728) secondary antibodies conjugated to horseradish peroxidase (HRP) were respectively purchased from GeneTex (Irvine, CA, USA) and Abcam (Cambridge, MA, USA). ARP101 (#A4433) was obtained from ApexBio Technology (Houston, TX, USA). All chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
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