The cells were lysed using EzRIPA buffer (ATTO, Tokyo, Japan). The lysate protein concentration was quantified by a Bradford assay (BioRad, Hercules, CA, USA) and measured using a BioTek Epoch Microplate Reader. Twenty micrograms of protein were subjected to 10%‐15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Amersham, GE Healthcare, Barcelona, Spain) using the electrophoretic method. The membrane was blocked by a PBS‐T solution which contained 5% skim milk for 60 minutes at room temperature. The primary antibodies, PINK1 (Novus Biologicals, Littleton, CO USA, #NB100‐493), phosphorylated AMPK (Cell Signaling, Boston, MA, USA, #2531), MDR1 (Santa Cruz biotechnology, CA, USA, #Sc‐55510) and β‐actin (Cell Signaling, Boston, MA, USA, #4967) were diluted 1:1000 in blocking solution (PBS‐T with 4% BSA) and incubated overnight at 4°C. The secondary HRP‐conjugated anti–rabbit (Santa Cruz Biotechnology) and anti–mouse (Santa Cruz Biotechnology) antibodies were used at a dilution of 1:1000 in blocking solution for 2 hours. Protein expression was detected by a chemiluminescence imaging system (ATTO) after spreading the Luminata Forte Western HRP Substrate (Millipore).
Phosphorylated ampk
Manufactured by Cell Signaling Technology
Sourced in United States
Phosphorylated AMPK is a protein that serves as a sensor of cellular energy status. It becomes activated when cellular AMP levels are high, indicating low energy status. Activated phosphorylated AMPK helps restore energy balance by inhibiting anabolic pathways and activating catabolic pathways.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (7 mentions)
Phosphorylated mtor, by Cell Signaling Technology (4 mentions)
Eif2α, by Cell Signaling Technology (2 mentions)
Nf κb p65, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Pink1, by Novus Biologicals (1 mentions)
Mdr 1, by Santa Cruz Biotechnology (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Epoch microplate reader, by Agilent Technologies (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Ac 15, by Abcam (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
Phosphorylated chk1, by Cell Signaling Technology (1 mentions)
Phosphorylated h2a x, by Cell Signaling Technology (1 mentions)
Mouse antibody against α tubulin, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ab128874, by Abcam (1 mentions)
19 protocols using phosphorylated ampk
1
Western Blot Analysis of Protein Targets
2
Quantitative AMPK Signaling Pathway Analysis
3
Immunoblotting Analysis of DNA Damage Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 22–24 hpf, 30–40 embryos per group were lysed, and 30 μg of protein was separated on a 12% SDS-PAGE gel, transferred onto nitrocellulose membrane and probed with rabbit antibodies against phosphorylated H2A.X (at residue Ser139), phosphorylated Chk1 (at residue Ser345), phosphorylated AMPK (at residue Thr172) (Cell Signaling, Beverly, MA), or p53 (AnaSpec, Fremont, CA) followed by a horseradish peroxidase (HRP)-conjugated antibody against rabbit IgGs (Santa Cruz Biotechnology, CA). The membrane was stripped and re-probed with a mouse antibody against α-tubulin (Sigma) followed by a HRP-conjugated antibody against mouse IgGs (Santa Cruz Biotechnology). SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) was used for detection.
4
Caerulein-Induced Pancreatitis Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caerulein (CER) (#HY-A0190) was purchased from MedChemExpress (Monmouth Junction, NJ). Palmitoleic acid (POA), CCK, L-Arginine (L-Arg), and Na taurocholate (NaT) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). BSA was purchased from Yeasen (Shanghai, China). JQ1, EX527 were purchased from Selleck Chemicals (Houston, TX, USA). Antibodies against microtubule associated protein 1 light chain 3 beta (LC3B) (#3868S), p62 (#23214S), ATG14 (#96752S), cathepsin B (#31718S), AMP activated protein kinase (AMPK) (#5831T), phosphorylated AMPK (#2535T), mammalian target of rapamycin (mTOR) (#2983T), phosphorylated mTOR (#5536T), β-actin (#3700s) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against Amylase (#sc46657) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against BRD4 (#ab128874), LAMP2 (#ab203224), syntaxin 17 (STX17) (#ab229646), cathepsin L (#ab6314) were purchased from Abcam (Cambridge, MA, USA). Antibody against CD45 (#550539) were purchased from BD Biosciences (Franklin, NJ, USA).
5
Tissue Collection and Analysis in Fasted Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fasted (5 hr) mice were anaesthetised with methoxyflurane at week 12, and liver and white adipose tissues were collected and fixed in 4% formaldehyde overnight before being incubated in 50% ethanol and embedded in paraffin. The tissues were then cut into 4 μm sections and stained with hematoxylin and eosin (CellCentral, UWA) before being visualized and photographed using a Nikon Eclipse TS100 microscope. Additional liver, white and brown adipose tissues, gastrointestinal tract, kidney, and skeletal muscle were collected and snap frozen in liquid nitrogen and stored at −80°C until analysis. White and brown adipose tissue protein expression of AMPK (Cell Signaling, USA), phosphorylated AMPK (Cell Signaling, USA), ACC (Cell Signaling, USA), phosphorylated ACC (Cell Signaling, USA), and UCP-1 (Cell Signaling, USA) were determined using western blot and normalised to actin (Sigma, USA), as previously described [7 (link)]. In a subset of mice (5/group), kidneys were also collected and stained with antisodium glucose cotransporter 2 (SGLT2, Santa Cruz Biotechnology) as previously described [10 (link)].
6
Western Blot Analysis of Silkworm Fat Body Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis for detection of proteins in the silkworm fat bodies was performed as previously described24 (link). Briefly, fat bodies isolated from the dorsolateral region of the larvae were rinsed in insect saline (10 mM Tris/HCl, 130 mM NaCl, 5 mM KCl, and 1 mM CaCl2) and then transferred to NP-40 lysis buffer (10 mM Tris/HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, 1% NP-40, 10 mM NaF, and 1 mM Na3VO4), and lysed by sonication using Sonifier 450 (Branson). The mixture was precipitated with 5% trichloroacetic acid, electrophoresed in a 12.5% polyacrylamide gel, and electroblotted onto a polyvinylidene difluoride membrane (Millipore), probed with antibody, and detected using Western Lightning (Perkin-Elmer Life Sciences). The following antibodies were used for immunoblot analysis: rabbit polyclonal antibodies to, phosphorylated Akt (Cell Signaling), phosphorylated JNK (Promega), phosphorylated AMPK (Cell Signaling) and β-actin (Cell Signaling). Quantification of the amount of phosphorylated JNK, phosphorylated Akt, or phosphorylated AMPK was performed by densitometric scanning with Image Gauge software. The relative amount of phosphorylated JNK or phosphorylated Akt or phosphorylated AMPK to β-actin was determined.
7
Oridonin and AMPK Pathway Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oridonin (C20H28O6) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary rabbit monoclonal antibodies (diluted at 1:1,000) against phosphorylated-LKB1, phosphorylated- AMPK, E-cadherin (E-Ca), N-cadherin(N-Ca) and β-actin were purchased from Cell Signaling Technology, Inc (Beverly, MA, USA). AICAR and Compound C, OE-LKB1
8
Molecular Mechanisms of AMPK Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TNF-α and 2-aminopurine (2-AP) were purchased from Sigma Chemical (St. Louis, MO, USA). AICAR and compound C (CC) were purchased from Calbiochem (San Diego, CA, USA). Antibodies against AMPK, phosphorylated-AMPK, β-actin, perilipin, hormone sensitive lipase (HSL), eIF2α, phosphosphorylated-eIF2α, PERK, and phosphorylated-PERK were purchased from Cell Signaling (Beverly, MA, USA).
9
Quantifying Skeletal Muscle Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein levels from skeletal muscle were measured by western blotting using tissue lysates (50 μg/lane) as described in our previous methods (Yano et al., 2020 (link)). In brief, the blots were incubated with their respective polyclonal antibodies, which included polyclonal rabbit Beta-actin (Cell signaling Technology, Beverly, MA), integrin αvβ5 (Abcam), phosphorylated AMPK, and non-phosphorylated AMPK (Cell signaling Technology, Beverly, MA) at a diluted concentration of 1:1000. The signals were then visualized using anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000). The results were visualized with Super Signal West Pico ECL chemiluminescence reagent (Thermo-Fisher Scientific). Densitometric analysis for the blots were completed using NIH Image J processing program.
10
Sirt1-mediated AMPK regulation in cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
miR-138 mimics and negative control were purchased from Guangzhou RiboBio, Co., Ltd. (Guangzhou, China). ShR-silent information transcriptional regulator (Sirt1) lentivirus and negative control were constructed by Hanbio Co., Ltd. (Shanghai, China). Rabbit anti-human Sirt1 polyclonal antibodies were purchased from Abcam plc. (Boston, MA, USA). Antibodies of AMPK, phosphorylated AMPK, E-cadherin, vimentin, mTOR, phosphorylated mTOR, LC3B and p62 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). TRIzol and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). TIANScript RT kit for reverse transcription and Quant One-Step qRT-PCR kit were purchased from Tiangen Biotech, Co., Ltd. (Beijing, China). Transwell assay kit was purchased from Corning Co. (Boston, MA, USA).
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!