The samples were taken from the ischaemic zone. The expression levels of myocardial ALDH2 (1:1000, Abcam, Cambridge, USA), CHOP [Cell Signaling Technology (CST); 1:1000], caspase-12 (CST, 1:1000), caspase-9 (CST, 1:1000), caspase-3 (CST, 1:1000), Bax (CST, 1:1000), Bcl-2 (CST, 1:1000), gp91phox (Abcam; 1:1000), iNOS (Abcam; 1:1000), p-AMPKα (CST, 1:1000), AMPKα (CST, 1:1000), mTOR (CST, 1:1000), p-mTOR (CST, 1:1000), p70s6k (CST, 1:1000), p-p70s6k (CST, 1:1000), mitochondrial OXPHOS complexes, and GAPDH (CST, 1:1000) were determined by immunoblotting [25 (link)]. The quantitative protein band density was assayed by ImageJ 1.37.
Ampkα
Manufactured by Abcam
Sourced in United States
AMPKα is a catalytic subunit of the AMP-activated protein kinase (AMPK) complex. AMPK is a sensor of cellular energy status and plays a role in the regulation of metabolism.
Automatically generated - may contain errors
Lab products found in correlation
P ampkα, by Abcam (4 mentions)
Gapdh, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
β actin, by Abcam (2 mentions)
Sirt1, by Abcam (2 mentions)
Gp91phox, by Abcam (1 mentions)
Caspase 12, by Abcam (1 mentions)
Aldh2, by Abcam (1 mentions)
P70s6k, by Abcam (1 mentions)
P p70s6k, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
Caspase 9, by Abcam (1 mentions)
P mtor, by Abcam (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Ab199970, by Abcam (1 mentions)
Ab37168, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Hrp conjugated goat anti mouse igg h l, by Wuhan Servicebio Technology (1 mentions)
12 protocols using ampkα
1
Myocardial Protein Expression Analysis
2
Western Blot Analysis of AMPK Pathway
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The primary antibodies were against the following: AMPKα (#5831, CST Inc., Danvers, MA, USA), phospho-AMPKα (#2535, CST Inc., p-AMPKα), serine–threonine kinase 11 (STK11) (ab199970, Abcam plc., Cambridge, CB, UK), phospho-STK11 (#3482, CST Inc., p-STK11), PKA (#4782, CST, Inc.), phospho-PKA (#9621, CST Inc., p-PKA), TSC2 (#4308, CST Inc.), phospho-TSC2 (#3617, CST Inc., p-TSC2), mTOR (#2792, CST Inc.), and phospho-mTOR (#2972, CST Inc.; p-mTOR). The reference protein was GAPDH (ab37168, Abcam plc.). The procedure was conducted according to a previous report (Chen et al., 2019 (link)). The primary antibodies were diluted with 5% BSA at 1:1,000 and incubated overnight at 4 °C. The density of bands was quantified by using BIO-RAD Gel Doc XR+ (Bio-Rad Laboratories, Hercules, CA, USA) and Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA), and the relative expression levels were normalized to the reference protein expression level.
3
Western Blot Analysis of Fibrotic Markers
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Total protein was extracted by RIPA lysis buffer (Beyotime Biotechnology, P0013B) supplemented with protease inhibitors (Merck, HY-K0010) and phosphatase inhibitors (Bimake, B15002). The following primary antibodies are used for Western blot: AMPKα (Abcam, ab32047), p-AMPKα (Cell Signaling Technology, 2535S), α-SMA (Proteintech, 23660-1-AP), Collagen I (Proteintech, 14695-1-AP), CTGF (Proteintech, 23936-1-AP), PAI-1 (Proteintech, 13801-1-AP), GAPDH (Proteintech, 60004-1-Ig), YAP (Proteintech, 13584-1-AP), p-YAP (Cell Signaling Technology, 13008). Horseradish peroxidase (HRP)-conjugated Goat Anti-Rabbit IgG (H + L) and HRP-conjugated Goat Anti-Mouse IgG (H + L) from Servicebio (GB23303, GB23301) were used as secondary antibodies.
4
Western Blot Analysis of GPR65, JNK, and AMPK
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The cells were lysed with RIPA buffer containing protease inhibitors (Beyotime, Shanghai, China). Total proteins were collected and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated with primary antibodies. After being washed, the membranes were then incubated with horseradish peroxidase–linked secondary antibodies. The antibodies used were as follows: GPR65 (1:1,000, Biorbyt), JNK (1:2,000; Santa Cruz, Dallas, TX, United States), phosphorylation JNK (1:2,000; Santa Cruz), AMPKα (Abcam, Cambridge, MA, United States), phosphorylation AMPKα(), GAPDH (Abcam), goat anti-rabbit immunoglobulin G (1:5,000; Abcam).
5
Antibodies for Protein Analysis
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Antibodies against the following proteins were obtained from Cell Signaling Technology (Danvers, MA, USA): ACC (1:1,000 for immunoblotting, #3662), phosphorylated (phospho-)ACC (Ser79) (1:1,000 for immunoblotting, #3661), AMPKα (1:1,000 for immunoblotting, #2532S), AMPKα2 (1:1,000 for immunoblotting, #2757S), phospho-AMPK (Ser172) (1:1,000 for immunoblotting, #2531S), GAPDH (1:1,000 for immunoblotting, #2118), S6 (1:1,000 for immunoblotting, #2217), phospho-S6 (1:1,000 for immunoblotting, #4858), β-actin (1:1,000 for immunoblotting, #4967), and normal rabbit IgG (5 μg/sample for immunoprecipitation, #2729); from Abcam (Cambridge, UK): AMPKγ1 (1:1,000 for immunoblotting, 5 μg/sample, for immunoprecipitation, ab32508), AMPKα1 (1:1,000 for immunoblotting, 1:1,000 for immunofluorescence, ab3759), dysferlin (5 μg/sample for immunoprecipitation, ab214078), GFP (1:1,000 for immunoblotting, ab290), red fluorescent protein (RFP) (1:1,000 for immunoblotting, ab62341); from Leica Biosystems (Nussloch, Germany): dysferlin (1:250 for immunoblotting, NCL-Hamlet); from Sigma (St. Louis, MO): dysferlin (1:250 for immunofluorescense, HPA017071); and from Medical and Biological Laboratories (Nagoya, Japan): RFP (5 μg/sample for immunoprecipitation, M208-3).
6
Western Blot Analysis of Apoptosis and Autophagy Markers
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The primary antibodies were purchased from Cell Signaling Technology (cleaved-Caspase 3 (Cat: 9664, 1:1000), PARP (Cat: 9542, 1:1000), AMPKα (Cat: 5831, 1:1000), Phospho-AMPKα (Thr172) (Cat: 4188, 1:1000)) and Abcam Trading (Shanghai) Company Ltd. (PLK1(Cat: ab17056, 1:1000), LC3Ⅱ (Cat: ab48394, 1:1000), P62 (Cat: ab56416, 1:1000)). The antibody against β-actin, which was used as the reference protein, was purchased from Sigma-Aldrich (Cat: A5441, 1:5000). The horseradish peroxidase- conjugated secondary antibodies Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Cat: 115-035-003) and Goat Anti-Rabbit IgG (H+L) (Cat: 111-035-003) were purchased from Jackson ImmunoResearch Laboratories, Inc.
7
Western Blot Analysis of Signaling Proteins
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The collected cells were lysed and resolved by 12% sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (MerckMinipore). The blots were probed with primary antibodies. Primary antibodies were obtained from Cell Signaling Technology and included antibodies against the following targets, Akt, p‐Akt S473, AMPKα, phosphor (p)‐AMPKα, and GAPDH, and SIGIRR were acquired from Abcam. The primary antibodies were detected using horseradish peroxidase‐conjugated goat anti‐rabbit antibodies (Invitrogen, A27036 1:10000 dilution) and further visualized using ECL reagents (Merck Millipore, WBKLS0500). ImageJ software was used to further quantify the band intensity in the image, including only the band intensity in the linear range.
8
Western Blot Analysis of Heart and Myocyte Lysates
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Heart and myocyte lysates were prepared as described (Hao P. et al., 2015 (link)). Proteins in lysates were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States), which were incubated with primary antibodies for PRCP (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, United States); LC3 (1:1,000), PINK1 (1:200), Parkin (1:200), COX IV (1:1,000), p-AMPK (1:1,000), AMPKα (1:1,000), p-Akt (1:500), or Akt (1:500; all from Abcam, Cambridge, MA, United States); or β-actin (1:1,000; Cell Signaling Technology, Danvers, MA, United States), followed by appropriate horseradish peroxidase-labeled secondary antibodies. The protein level of PRCP was normalized to that of β-actin as an internal control, the levels of PINK1 and Parkin were normalized to that of COX IV, and the levels of phospho-proteins were normalized to those of total proteins.
9
AMPK and Antioxidant Pathway Analysis
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GA was obtained from Tokyo Chemical Industry Co. (TCI, Tokyo, Japan). A reactive oxygen species assay kit was purchased from Beyotime (Jiangsu, China). AMPKα, p-AMPKα, SIRT1, PGC-1α, TGF-β1, and α-SMA antibodies were purchased from Abcam. β-actin was purchased from Bios (Beijing, China).
10
Protein Extraction and Western Blot Analysis
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For protein extraction, cells transfected as aforementioned or retinal tissues were lysed in RIPA lysis buffer (Beyotime) with freshly added protease inhibitor PMSF (Beyotime). The samples were lysed at 4 ° C for 15 min, centrifuged at 12000r/min at 4 ° C for 15 min. The protein concentration was measured by BCA kit (Beyotime), and the proteins were denatured at 97 ° C for 10 min. Next, SDS-PAGE electrophoresis was performed and the proteins were transferred to PVDF membrane. 50 g/L skim milk powder was used to block the membranes for 60 min at room temperature. Then the membranes were incubated with the primary antibodies in a shaker at 4 ° C overnight (LC3B, 1:600 dilution; p62, 1:800 dilution; p-AMPKα, 1:1000 dilution; AMPKα, 1:1500 dilution; SIRT1, 1:1200 dilution; Abcam, USA). The next day, the membranes were washed three times and incubated with the secondary antibody in a shaker at 4 °C for 2 h. Finally, the membranes were treated with ECL luminescent solution (Beyotime) in the dark. ImageJ (version 1.48; National Institutes of Health) software was used to quantify protein expression.
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