The liver tissue of mice (0.1 g) was added to the mixture including RIPA lysis buffer (1 mL) and phosphatase inhibitor cocktail (10 μL), homogenized by a glass homogenizer (2 mL), incubated for 20 min in ice water, and centrifuged at 14000 rpm for 10 min to obtain the supernatant, which was mixed with loading buffer (5x) and boiled for 10 min. The obtained protein solution was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (0.45 μm), which were blocked with 5% (w/v) BSA in TBST at room temperature for 2 h. The enclosed membrane was incubated with primary rabbit antibodies (anti-p-AMPKα, 1 : 1000; anti-GAPDH, 1 : 1000, Cell Signaling Technology Inc., Boston, USA) overnight at 4°C and then employed with peroxidase-conjugated goat anti-rabbit as secondary antibodies (1 : 5000) (Absin Bioscience Inc., Shanghai, China) at room temperature for 1 h. Protein bands were visualized using the ECL Western Blotting System (GE Healthcare Life Sciences) and band density quantified using an AlphaImager imaging system (Alpha Innotech Corporation).
Anti p ampkα
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-p-AMPKα is a primary antibody that specifically detects phosphorylated AMPKα (Thr172). AMPK is a cellular energy sensor that plays a crucial role in regulating cellular metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Anti ampkα, by Cell Signaling Technology (15 mentions)
Anti mtor, by Cell Signaling Technology (4 mentions)
Anti p mtor, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti p p70s6k, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti p akt, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated goat antirabbit or antimouse secondary antibody, by Cytiva (2 mentions)
Anti p erk, by Santa Cruz Biotechnology (2 mentions)
Anti erk, by Santa Cruz Biotechnology (2 mentions)
Anti ampkα1, by Cell Signaling Technology (2 mentions)
Anti foxm1, by Santa Cruz Biotechnology (2 mentions)
Anti enos, by Santa Cruz Biotechnology (2 mentions)
Anti penosser1177, by Santa Cruz Biotechnology (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Anti pcna, by Santa Cruz Biotechnology (2 mentions)
25 protocols using anti p ampkα
1
Western Blot Analysis of Mouse Liver Proteins
2
Immunohistochemistry of Cochlear AMPK
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Cochlear paraffin sections were deparaffinized with xylene and rehydrated in ethanol at graded concentrations. Rinsed in 3% hydrogen peroxide for 10 minutes and block solution for 30 minutes at room temperature. Then immersed in the following primary antibodies in a wet box at 4 °C overnight: anti-p-AMPKα (2535, Cell Signaling Technology Inc., Beverly, MA, USA) at 1:100, anti-AMPKα1 (ab32047, Abcam, Cambridge, England, UK) at 1:200 and anti-AMPKα2 (ab3760, Abcam, Cambridge, England, UK) at 1:100, respectively. Then incubated in the secondary antibodies HRP-labeled goat anti-rabbit IgG (A0208, Beyotime, Shanghai, CN) for 30 minutes in darkness at room temperature. The peroxidase reaction was visualized by using diaminobenzidine (DAB) reagent. The slides were finally dehydrated, cleared and mounted with coverslips.
3
Immunostaining and Western Blotting Antibodies
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Anti-dystrophin (ab15277, 1:100 for immunostaining) and anti-VDAC1 (ab15895, 1:1000 for western blotting hereinafter) antibodies were purchased from abcam; anti-pY (4G10, 1:1000) antibody was from Upstate; and the others, anti-p-Akt (9271, 1:1000), anti-Akt (9272, 1:1000), anti-p-AMPKα (2531, 1:1000), anti-AMPKα (2532, 1:1000), anti-p-AS160 (4288, 1:1000), anti-AS160 (2447, 1:1000), anti-p-FoxO1 (9461, 1:1000), anti-FoxO1 (9454, 1:1000), anti-FoxO4 (9472, 1:1000), anti-GAPDH (2118, 1:5000), anti-LC3B (2775, 1:1000), anti-p-mTOR (2971, 1:1000), anti-mTOR (2972, 1:1000), antibodies were from Cell Signaling. Secondary antibody for immunostaining (Alexa 594 Goat Anti-Rabbit; A11037, 1:400) was purchased from Invitrogen, and those for western blotting (HRP-linked Anti-Mouse [from Sheep] and Anti-Rabbit [from donkey]; NA931 and NA934 respectively, both 1:2000) were from GE Healthcare Life Sciences.
4
Immunoblotting Analysis of Protein Signaling
5
Western Blot Analysis of Endothelial Proteins
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Equal amounts of proteins (40 μg) were separated by stain-free SDS-polyacrylamide gel electrophoresis (7.5%), and then transferred to mini low fluorescence polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hemel Hempstead, UK). The blots were visualised using Chemidoc MP Imaging system (Bio-Rad Laboratories), then the membrane was blocked with 3.5% (w/v) bovine serum albumin in TBS-T (10 mM Tris pH8, 150 mM NaCl, 0.1% Tween 2.0) for an hour at room temperature before exposure to primary antibody (anti-eNOS, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA; anti-peNOSser1177, 1:200, Santa Cruz Biotechnology; anti-AMPKα, 1:500, Cell Signalling (Hitchin, UK); anti-pAMPKα, 1:500, Cell Signalling; 4 °C, overnight) and then horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:10 000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for an hour at room temperature. Chemiluminescence was performed with Clarity Western ECL Substrate (Bio-Rad Laboratories) and detected using Chemidoc MP Imaging system. Blots were quantified using ImageLab 5.2.1. (Bio-Rad Laboratories) software and protein expression was normalised to the total protein of the sample as previously described.34 (link)
6
Western Blot Analysis of Signaling Proteins
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Cells were harvested in 1× RIPA Buffer lysis buffer and protein concentrations were determined as previously described. Approximately 50 μg of protein was loaded and separated by SDS-PAGE, transferred to a PVDF membrane (Millipore, Danvers, MA, USA), and incubated with the following primary antibodies: anti-p-AMPKα (#2535), AMPKα (#2532), p-AKT (#9271), total AKT (#9272), phos-pP44/42 (#9101), P44/42 (#9102), p-STAT3 (#9131), total STAT3 (#9132), and N-cadherin (#4061), which were all purchased from Cell Signaling Technology (CST) Danvers, MA, USA, anti-E-cadherin(sc-7870), integrin β1 (sc-9970), vimentin (sc-373717), smooth muscle actin (SMA; sc-53142), CD44 (sc-18849), CD34 (sc-18917), and GAPDH (sc-47724) antibodies, which were all purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and anti-β-actin (#A1978) antibody, which was purchased from Sigma-Aldrich, St. Louis, MO, USA.
7
Protein extraction and Western blot analysis
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Total, cytoplasmic, and nuclear proteins of cells were extracted and assessed by Western blot analysis with antibodies using a standard protocol described previously [26 (link),27 (link)]. We used the following antibodies: anti-PLCɛ (Santa Cruz); anti-PCNA, anti-Cyclin D1, anti-β-actin, anti-p-AMPKα, anti-total-AMPKα, ACLY, ACACA, SCD1 (Cell Signaling Technology), anti-SREBP-1, anti-H3 (Abcam), and FASN (Sigma).
8
Protein Extraction and Western Blot Analysis of Liver Samples
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Liver tissues and primary hepatocytes were harvested and lysed in RIPA buffer (#G2002; Servicebio, Wuhan, China) with the protein concentrations measured by the Pierce™ BCA Protein Assay kit (#23225; ThermoFisher Scientific) [33 (link)–35 (link)]. Next, equal amounts of total proteins were separated by SDS-PAGE and transferred onto PVDF membranes that were then blocked with 5% skimmed milk at room temperature for 1 h and probed with indicating primary antibodies at 4°C overnight. The primary antibodies against NRF2 (#ab92946), β-actin (#ab8226), phospho-NF-κB p65 (p-p65, #ab76302), and total-p65 (t-p65, #ab32536) were purchased from Abcam, while anti-p-AMPKα (#50081) and anti-t-AMPKα (#5832) were purchased from Cell Signalling Technology (Beverly, MA, USA). Anti-PDE4D (#12918-1-AP) was purchased from Proteintech (Rosemont, IL, USA). On the second day, the membranes were incubated with the HRP-conjugated secondary antibodies at room temperature for 1 h. Subsequently, the protein bands were visualized by LumiGLO chemiluminescent substrate and quantified using Image Lab software.
9
Immunoblotting Analysis of Protein Signaling
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Proteins in cell lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene-difluoride membranes. The membranes were blotted with 5% skimmed milk and subsequently probed overnight at 4°C with primary antibodies specific for anti-p-AMPKα, anti-AMPKα1, anti-AMPKα, anti-p-AKT, anti-AKT, anti-p-p70S6K, anti-p-mTOR, anti-mTOR (Cell Signaling, Beverly, MA), anti-p-ERK, anti-ERK, anti-FOXM1 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), and anti-β-actin (Sigma-Aldrich, St Louis, MO) and then incubated with horseradish peroxidase conjugated goat antirabbit or antimouse secondary antibody (Amersham, Uppsala, Sweden). Immunodetection was performed with enhanced chemiluminescence reagent solution (Amersham ECL) and visualized by medical X-ray film.
10
Reagents and Antibodies for Cell Signaling Analysis
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Dimethyl sulfoxide (DMSO), bicinchoninic acid (BCA), trichloroacetic acid (TCA), sulforhodamine B (SRB), and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsin-EDTA solution (1X), antibiotic-antimycotic solution (100X) and phosphate-buffered saline (PBS) (1X) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-p-AMPKα, anti-AMPKα, anti-p-ACC, anti-ACC, anti-p-mTOR, anti-mTOR, anti-p-4EBP1, anti-4EBP1, anti-p-eIF4E, anti-eIF4E, anti-p-P70S6K1, anti-P70S6K1, anti-p-RPS6, anti-RPS6, anti-rictor, anti-p-Akt, anti-Akt, and anti-E-cadherin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-PKC-α, anti-p-Rac1, anti-Rac1, anti-PCNA, anti-β-actin, and all secondary antibodies were purchased from Santa Cruz Biotechnology. (Santa Cruz, CA, USA). Anti-p-rictor was purchased from Millipore (Temecula, CA, USA). Anti-p-PKC-α, anti-F-actin, and anti-Ki-67 were purchased from Abcam (Cambridge, MA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA) and rapamycin was purchased from Tocris Bioscience (Bristol, UK).
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