Protein was extracted using RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein (20 µg) was separated by 8% SDS-PAGE and transferred on to PVDF membrane. The membranes were incubated with antibodies of ChREBP (1:1,000, Abcam, cat. no. ab92809), fatty acid synthase (FASN; 1:500, Wuhan Boster Biological Technology, Ltd., cat. no. PB0909), acetyl-CoA carboxylase (ACC; 1:500, Wuhan Boster Biological Technology, Ltd., cat. no. BM4414), AMPK (1:1,000, Cell Signaling Technology, Inc., cat. no. 2532), phosphorylated (p)-AMPK (Thr172; 1:1,000, Cell Signaling Technology, Inc., cat. no. 2535), AKT (1:1,000, Cell Signaling Technology, Inc., cat. no. 9272), p-AKT (Ser473; 1:1,000, Cell Signaling Technology, Inc., cat. no. 9271), GAPDH (1:2,000, Wuhan Boster Biological Technology, Ltd., cat. no. BM1623) and FOXO1 (1:500, Wuhan Boster Biological Technology, Ltd., BM4249) overnight at 4°C. Then, membranes were incubated with rabbit anti-mouse lgG (cat. no. BA1048) and goat anti-rabbit lgG antibodies (cat. no. BA1039; both from Wuhan Boster Biological Technology, Ltd.). Finally, the results were detected by ECL reagents (Beyotime Institute of Biotechnology) and semi-quantified by densitometry using the Tanon 5200 automatic chemiluminescent imaging system (Tanon Science and Technology Co., Ltd).
Phosphorylated ampk thr 172
Manufactured by Cell Signaling Technology
Sourced in United States
Phosphorylated AMPK (Thr 172) is a laboratory product offered by Cell Signaling Technology. It is a protein that has been phosphorylated at the threonine 172 residue. AMPK is a cellular energy sensor that plays a crucial role in regulating metabolism and cellular homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
P akt ser473, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Ripa lysis buffer, by Wuhan Boster Biological Technology (1 mentions)
Chrebp, by Abcam (1 mentions)
Gapdh, by Wuhan Boster Biological Technology (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
P ulk1 ser757, by Cell Signaling Technology (1 mentions)
Beclin 1, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
P stat3 tyr705, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Phosphorylated stat3 at tyr705, by Cell Signaling Technology (1 mentions)
P stat3 ser727, by Cell Signaling Technology (1 mentions)
Supersignal west pico kit, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Phosphorylated p38 mapkthr180 tyr182, by Cell Signaling Technology (1 mentions)
7 protocols using phosphorylated ampk thr 172
1
Western Blot Analysis of Lipogenic Proteins
2
Autophagy-related Protein Expression Analysis
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Antibodies against the following proteins were used: Sequestosome 1 (p62; 1:1,000; cat. no. ab109012; Abcam), Beclin1 (1:1,000; cat. no. ab210498; Abcam), cleaved caspase-3 (1:1,000; cat. no. AB3623; Sigma-Aldrich; Merck KGaA), cleaved caspase-9 (1:1,000; cat. no. AB3629; Sigma-Aldrich; Merck KGaA), microtubule-associated protein light chain 3 A/B (LC3A/B; 1:1,000; cat. no. 4108; Cell Signaling Technology, Inc.), AMPK (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), phosphorylated (p)-AMPK (Thr172) (1:1,000; cat. no. 2535; Cell Signaling Technology, Inc.), Unc-51 like autophagy activating kinase 1 (ULK1; 1:1,000; cat. no. 6439; Cell Signaling Technology, Inc.), p-ULK1 (Ser757) (1:1,000; cat. no. 14202; Cell Signaling Technology, Inc.) and β-actin (1:5000; cat. no. sc-47778; Santa Cruz, Biotechnology. Inc.). Horseradish peroxidase-conjugated secondary antibodies (anti-mouse/rabbit IgG) (1:5,000; cat. nos. 7076 and 7074; Cell Signaling Technology, Inc.) were used.
3
Western Blot Analysis of Cardiac Proteins
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Equal protein amounts from isolated cardiomyocytes, rat heart, and isolated mitochondria were resolved by 8-12% SDS-PAGE and transferred to polyvinylidene fluoride membrane for immunoblot analysis, as previously described [26 (link)]. Membranes were blocked with 5% nonfat milk in Tris-Buffered Saline- (TBS-) Tween and were incubated with primary antibodies overnight at 4°C at the following dilutions: AMPK, phosphorylated AMPK (Thr172), STAT3, phosphorylated STAT3 at Tyr705 (p-STAT3 Tyr705), Bax, Bcl2, caspase-3, and cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) 1 : 1000; phosphorylated STAT3 at Ser727 (p-STAT3 Ser727) (Cell Signaling Technology, Beverly, MA) 1 : 500. After washing with TBS-Tween, immunoreactive bands were visualized by an enzymatic chemiluminescence method and quantified with Quantity One image software.
4
Protein Signaling in Exercise Adaptation
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Western blot analysis was only performed for pre- and postexercise time periods due to limited sample availability. Homogenates used for the citrate synthase activity assay were centrifuged for 15 min at 10,000 × g at 4°C, the supernatant (lysate) was collected, and protein content was analyzed (ThermoFisher). Equal amounts of total protein (15 µg) were used in precast 4–20% Mini-PROTEAN TGX gels (Bio-Rad Laboratories). Proteins were transferred to polyvinylidene fluoride membranes and exposed to phosphorylated p38 MAPKThr180/Tyr182 and phosphorylated AMPKThr172 (Cell Signaling Technology) primary antibodies at 4°C overnight. Labeling was performed by using secondary antibody (anti-rabbit IgG conjugate with HRP; Cell Signaling Technology) and chemiluminescent reagents (Super Signal, West Pico Kit; Pierce Biotechnology). Blots were quantified by using a phosphoimager (ChemiDoc XRS; Bio-Rad) and Image Lab software (Bio-Rad). GAPDH was used to confirm equal protein loading per well. All data are presented as fold changes relative to the pre-exercise time period.
5
AMPK Activation by Seaweed Extracts in C2C12 Cells
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To determine the alteration of AMPK activation by seaweed extracts in C2C12 cells, Western blotting analysis was performed as described previously [19 (link)]. Briefly, the extracted proteins (30~50 μg/24 μL) from the C2C12 myotubes treated with either control or seaweed extracts were quantified. Equal amounts of proteins were loaded and separated by SDS–PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with the indicated antibody and horseradish peroxidase-coupled anti-species antibodies. The antibodies used were the following; phosphorylated AMPK (Thr172, Cell Signaling, Beverly, MA, USA, 1:1000), AMPK (Cell Signaling, Beverly, MA, USA, 1:1000), GAPDH (Cell Signaling, Beverly, MA, USA, 1:1000). Proteins were visualized using Chemidoc (Bio-Rad, Hercules, CA, USA) and quantified using Image J (National Institutes of Health, Bethesda, MD, USA).
6
Western Blot Analysis of Cellular Signaling
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Cell lysate was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The protein was transferred to a nitrocellulose membrane and was hybridized with antibody against integrin β-3 (Catalog Number: #13166), AMPK (#2532), phosphorylated AMPK (Thr 172) (#2535), phosphorylated p53 (Ser 15) (#9284) (Cell Signaling, Danvers, MA, USA), plasminogen activator inhibitor (#ab20562), a disintegrin and metalloproteinase with thrombospondin motifs 5 (#ab41037) (Abcam), IGFBP3 (#sc-9028), CARP (sc-30181), β-2 adrenergic receptor (#sc-569) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated H2AX (Ser 139) (#613401) (BioLegend, San Diego, CA, USA), p53 (Ab-6, Clone DO-1) and tubulin-α (Clone DM1A) (Thermo Fisher Scientific) as a control followed by an appropriate second antibody. The membranes were developed with the ECL system (GE Healthcare, Buckinghamshire, UK).
7
Western Blot Analysis of Apoptosis and AMPK
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Western blot analysis was performed as described previously [26] . In brief, the protein concentration was measured using a bicinchoninic acid protein assay kit (YTHXBio, China). The extracted proteins were then separated by electrophoresis using a 12% sodium dodecyl sulphate polyacrylamide gel. The proteins were transferred to polyvinylidene uoride membranes (Millipore, USA) and blocked using Tris-buffered saline with Tween buffer containing 3% BSA. The membranes were incubated with primary antibodies against Bax (1:500), phosphorylated AMPK (Thr 172) (1:1000), total AMPK (1:5000) (all from Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (1:5000; Abcam), and GADPH (1:10000; YTHXBio, China) at 4°C overnight. The membranes were washed three times and then incubated with the horseradish peroxidase-conjugated secondary antibody (1:10,000) at 37°C for 40min. Finally, the bands were detected by an enhanced chemiluminescence kit (Millipore, USA) and the results were quanti ed using Total Lab Quant V11.5 (Newcastle upon Tyne, UK). The levels of Bcl-2/Bax (anti-/pro-apoptotic) proteins and AMPK-p/AMPK proteins were also normalized to that of GAPDH.
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