The methods used for Western blotting have been described previously [2 (link),22 (link)]. Proteins were separated by electrophoresis and transferred to a polyvinylidene fluoride membrane (Merck KGaA) via a wet transfer system. The membranes were then maintained overnight at 4 °C with the following primary antibodies: anti-total AMPK, anti-phosphorylated AMPK (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin (AbFrontier, Seoul, South Korea). The membranes were then maintained with an anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Antibody-antigen complexes were visualized using the EzWestLumi Plus Detection Kit (ATTO Corporation, Tokyo, Japan). Luminescent images were obtained using a LuminoGraph II Imaging System (ATTO Corporation).
Anti phosphorylated ampk
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phosphorylated AMPK is a laboratory reagent used to detect and study the phosphorylated form of the AMP-activated protein kinase (AMPK) enzyme. AMPK is a crucial cellular energy sensor that becomes activated when cellular energy levels are low, triggering metabolic pathways to restore energy balance. The anti-phosphorylated AMPK reagent can be used to analyze AMPK activation and signaling in various biological samples and experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (5 mentions)
Anti ampk, by Cell Signaling Technology (5 mentions)
Anti αampk, by Cell Signaling Technology (4 mentions)
Anti phosphorylated akt, by Cell Signaling Technology (3 mentions)
Anti phosphorylated mtor, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Horseradish peroxidase conjugated anti rabbit igg secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Anti total ampk, by Cell Signaling Technology (2 mentions)
Anti flag antibody, by Merck Group (2 mentions)
Anti sirt1, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti beclin 1, by Cell Signaling Technology (2 mentions)
Anti bax, by Cell Signaling Technology (2 mentions)
Anti p62, by Cell Signaling Technology (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Antiphosphorylated akt ser473, by Cell Signaling Technology (2 mentions)
Anti p38, by Cell Signaling Technology (2 mentions)
Anti α tubulin, by Cell Signaling Technology (2 mentions)
Luminograph 2 imaging system, by ATTO Corporation (1 mentions)
15 protocols using anti phosphorylated ampk
1
Western Blotting Protocol for AMPK Quantification
2
Quantifying AMPK Phosphorylation and Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phosphorylation and protein levels of αAMPK were determined as described51 –54 . Western blot analyses were performed with anti-phosphorylated-AMPK (Cell Signaling Technology #2535) and anti-αAMPK (Cell Signaling Technology #2532) antibodies. Protein levels of AdipoR were analyzed by western blotting, using an anti-Flag antibody (Sigma-Aldrich F1804).
3
Quantifying AMPK Phosphorylation and Protein Levels
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted using the lysis buffer containing 25 mM hydroxyethylpiperazine ethane sulfonic acid (HEPES), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA, 10 mM NaF, 2 mM Na VO, and a protease inhibitor cocktail (Roche Korea, Seoul, Republic of Korea). Proteins were separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Merck KGaA, Darmstadt, Germany) using a wet transfer system. Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-total AMPK, anti-phosphorylated AMPK, anti-total Akt, anti-phosphorylated Akt (Cell Signaling Technology, Beverly, MA, USA), PTP-MEG2 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (AbFrontier, Seoul, Republic of Korea). Membranes were then probed with an anti-rabbit-IgG-horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Antibody–antigen complexes were detected using enhanced chemiluminescence (ECL) reagents (GE Healthcare Korea, Songdo, Republic of Korea).
5
C2C12 Cell Lysis and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C2C12 cells were lysed in 20 mM Tris-HCl (pH 7.4), 5 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, and 1% NP-40 buffer supplemented with a protease inhibitor (1 µg/µL aprotinin, 1 µg/µL leupeptin, and 1 mM PMSF). Cell lysates were sonicated on ice twice for 15 seconds each time, and debris was removed by centrifugation (15,000 g) for 30 minutes at 4℃. Proteins (10 to 20 µg) were separated on a sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked with 5% skim milk in Tween-20-Tris-buffered saline (TBS-T) for 1 hour at room temperature, followed by incubation with primary antibody overnight at 4℃. The primary antibodies used were a total OXPHOS antibody cocktail (MitoScience, San Francisco, CA, USA), anti-IRS-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-pIRS-1 (Cell Signaling, Danvers, MA, USA), anti-Akt (Cell Signaling), anti-pAkt (Cell Signaling), anti-adenosine monophosphate (AMP)-activated protein kinase (AMPK; Cell Signaling), anti-phosphorylated AMPK (Cell Signaling), and anti-γ-tubulin (Sigma-Aldrich Co.). The secondary antibodies were anti-rabbit or anti-mouse immunoglobulin G.
6
Antibody-based Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies used for Western blotting are listed: anti-GAPDH (MAB374, Merck Millipore, Billerica, MA, USA), anti-alpha SMA (ab5694, Abcam, Cambridge, UK), anti-vimentin (5741, Cell Signaling, Beverly, MA, USA), anti-phosphorylated AMPK (2535, Cell Signaling), anti-AMPK (2532, Cell Signaling), anti-phosphorylated mTOR (5536, Cell Signaling), anti-mTOR (ab32048, Abcam) and anti-collagen I (sc-166865, Santa Cruz, CA, USA). The chemicals used were PDGF-bb (520-BB, R&D System, Minneapolis, MN, USA), PDGF-bb(520-BB, R&D System) and n-butylidenephthalide (ALFA, Derbyshire, UK). For the in vitro experiments, 10% (w/v) n-BP/DMSO was prepared as the stock.
7
Metformin's effects on OC cell lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four OC cell lines, SKOV3, IGROV1, CAOV3 and OVCAR3, were used for these experiments. SKOV3 cells were grown in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin under 5% CO2. IGROV1, CAOV3 and OVCAR3 cells were maintained in RPMI 1640 containing 10% FBS, 100 units/ml penicillin and 100 ug/ml streptomycin. Metformin, MTT dye, RNase A and anti-α-tubulin antibody were purchased from Sigma (St. Louis, MO). The anti-phosphorylated-AMPK, anti-pan-AMPK, anti-phosphorylated-S6 and anti-pan-S6 antibodies as well as the caspase-3 ELISA kit were purchased from Cell Signaling (Beverly, MA). The Annexin V FITC Kit was purchased from BioVison (Mountain View, CA). The ChemoTx® Invasion Kit was from NeuroProbe (Gaithersburg, MD). Enhanced chemiluminescence Western blotting detection reagents were purchased from Amersham (Arlington Heights, IL). All other chemicals were purchased from Sigma.
8
Western Blotting of Apoptosis and Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as previously described [16 (link)]. The primary antibodies used were as follows: anti-cleaved-caspase-3, anti-Bcl-2, anti-Bax, anti-phosphorylated-AMPK, anti-AMPK, anti-phosphorylated-Akt, anti-Akt, anti-LC3B, anti-p62, anti-Beclin1, anti-ATG5, anti-ATG7, anti-Acetylation, anti-Lamin B and anti-SIRT1 antibody from Cell Signaling Technology, Inc. (Danvers, MA). The anti-β-tubulin1 antibody was from Boster Biological Technology (Wuhan, China). The secondary antibodies were provided by Boster Biological Technology.
9
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phenylephrine (PE) was purchased from Tokyo Chemical Industry (P0398). Hispidulin (SML0582), Dimethyl sulfoxide (DMSO) (D2650), and EX-527 (E7034) were obtained from Sigma-Aldrich (St. Louis, USA). With the purpose of detecting specific proteins by Western blotting, the following primary antibodies, obtained from Cell Signaling Technology (Danvers, MA, USA), were applied: anti-AMPK (Thr172) (#2531), antiphosphorylated AMPK (#2532), anti-Sirt1(D1D7) (#5490) antibody, anti-phosphorylated p38 (Thr180/Tyr182) (#4511), anti-p38 (#8690), anti-phosphorylated Akt (Ser473) (#4060), anti-Akt (#9272), anti-phosphorylated JNK1/2 (Thr183/Tyr185) (#4668), anti-JNK1/2 (#9252), anti-Tom20 (D8T4N) (#42406), anti-OPA1 (D6U6N) (#80471), anti-Mitofusin2 (D1E9) (#11925), and anti-GAPDH (#2118) antibodies. Anti-PGC-1α antibody (66369-lg) and secondary antibodies, including goat anti-rat IgG (H+L), horseradish peroxidase (HRP) conjugate (SA00001-15), goat anti-mouse IgG (H+L), and HRP conjugate (SA00001-1), were obtained from Protein-tech Group (Wuhan, China). The anti-vinculin antibody was purchased from Sigma-Aldrich (V9264).
10
Antibody Panel for Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-SHP-1 was purchased from Abcam. Anti-AMPK, anti-phosphorylated-AMPK, anti-ZAP70, anti-phosphorylated-ZAP70, anti-mTOR, anti-phosphorylated-mTOR, anti-p70 S6 kinase, and anti-phosphorylated-p70 S6 kinase antibodies were acquired from Cell Signaling Technology.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!