Cell Signaling Antibodies used at 1:1000 in 5% BSA in TBS-T: phospho-AMPKα1/2 Thr172 (#2535), AMPK α1/2 (#2532), AMPKα2 (#2757), phospho-ACC Ser79 (#3661), ACC (#3662), phospho-Raptor Ser792 (#2083), Raptor (#2280). Mouse monoclonal hAMPKα2 (#MAB2850) was purchased from R&D Systems. Flag M2 agarose resin (A2220), β-actin (A5441), and Flag M2 monoclonal (F1804) were purchased from Sigma. Phenformin hydrochloride was obtained from Sigma (P7045) and dissolved in SILAC DMEM (Thermo scientific) with 10% Dialyzed FBS (GIBCO-Life Technologies). Pierce High pH Reverse-Phase Peptide Fractionation Kit was purchased from Thermo Scientific. Acclaim PepMap 75 μm x 2 cm C18 trap columns (#164535) and Acclaim PepMap RSLC 75 μm x 50 cm analytical columns (#164942) were also purchased from Thermo Scientific
Ampkα1 2
Manufactured by Cell Signaling Technology
Sourced in China, United States
AMPKα1/2 is a recombinant protein that represents the catalytic subunits of the AMP-activated protein kinase (AMPK) complex. AMPK is a cellular energy sensor that plays a key role in regulating cellular metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Ampkα2, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Phenformin hydrochloride, by Merck Group (1 mentions)
Silac dmem, by Thermo Fisher Scientific (1 mentions)
Phospho acc ser79, by Cell Signaling Technology (1 mentions)
Dialyzed fbs, by Thermo Fisher Scientific (1 mentions)
Phospho raptor ser792, by Cell Signaling Technology (1 mentions)
Flag m2 agarose resin, by Merck Group (1 mentions)
Flag m2 monoclonal, by Merck Group (1 mentions)
Pierce high ph reverse phase peptide fractionation kit, by Thermo Fisher Scientific (1 mentions)
Phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Egr 1, by Santa Cruz Biotechnology (1 mentions)
Edta free protease, by Thermo Fisher Scientific (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
P ampkα thr172, by Cell Signaling Technology (1 mentions)
P acc ser79, by Cell Signaling Technology (1 mentions)
Nadph oxidase 4, by Abcam (1 mentions)
6 protocols using ampkα1 2
1
Western Blot Analysis of AMPK Signaling
2
Immunoblot Analysis of Protein Signaling Pathways
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Immunoblot analysis was performed as previously described [73 (link)]. Briefly, the cells were lysed in modified RIPA buffer containing 50 mM HEPES, 0.3% NP-40, 75 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, and EDTA-free protease (Thermo Fisher Scientific, cat. no. 88266) and phosphatase inhibitors (Thermo Fisher Scientific, cat. no. 88667) on ice. 35 ug of proteins were resolved using 4%–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, cat. no. 4561096) and transferred to PVDF membranes (Bio-Rad, cat. no. 1620177). The transferred blot was blocked with 5% BSA for 30 min in TBS containing 0.05% Tween-20 at room temperature for 30 min. Then the membranes were immunoblotted with primary antibody Egr1 (Santa Cruz, cat. no. sc-189; 1:500), phospho-AMPKα (Thr172) (Cell Signaling, cat. no. 2535; 1:1,000), AMPKα 1/2 (total) (Cell Signaling, cat. no. 2532; 1:1,000), AMPKα2 (total) (Cell Signaling, cat. no. 2757; 1:1,000), and β-tubulin (Cell Signaling, cat. no. 2146; 1:1,000) at 4°C overnight.
3
AMPK and NOX4 Signaling in Renal Tissue
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Renal arterial tissue incubated 30 min with A769662 or vehicle were snap frozen in liquid nitrogen, homogenized and lysed in buffer containing Tris-HCl (pH 7.5) 50 mmol/L, EGTA 1 mmol/L, EDTA 1 mmol/L, Triton X-100 1% vol/vol, sodium orthovanadate 0.1 mmol/L, sodium fluoride 50 mmol/L, sodium pyrophosphate 5 mmol/L, sucrose 0.27 mol/L and protease inhibitor cocktail. The protein concentration was determined by Bradford assay (Bio-Rad Laboratories, S.A, Madrid, Spain). Protein lysates (7 μg) were subjected to 6,5% SDS-PAGE, electrotransferred on a polyvinylidene fluoride membrane and probed with the following primary antibodies: pACC Ser79 (1:1000; ref 3661), pAMPKα Thr172 (1:1000; ref 2535), AMPKα1/2 (1:1000; ref 2532) from Cell Signaling Technology (Leiden, The Netherlands); NADPH oxidase 4 (1:500; ref ab154244) from Abcam (Cambridge, UK); β-actin (1:5000; ref. A5316) from Sigma (St. Louis, MO, USA) [4 ,32 (link)]. The blots were visualized using enhanced chemiluminescence and quantified by densitometry with ImageJ free software. Values were expressed in relation to β-actin protein levels.
4
Transforming Growth Factor-β1 Signaling Pathway
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Recombinant human TGF-β1 was purchased from R&D systems (Minneapolis, MN, USA). FBZ was purchased from Wuxi AppTec (Wuxi, China). Nocodazole and colchicine were purchased from MCE (Shanghai, China). Bleomycin sulfate was purchased from Hisun Pharm (Taizhou, China). Antibody to collagen I, Fibronectin, N-cadherin, phosphor-Thr172 AMPK, AMPKα1/2, phosphor-Thr389 P70S6K, P70S6K, α-Tubulin, acetylated (K40) α-Tubulin, and Alexa Fluor® 488-conjugated anti-rabbit IgG were obtained from Cell Signaling Technology (Shanghai, China). Goat anti-rabbit and anti-mouse IgG with horseradish peroxidase (HRP) conjugate as well as an antibody to α-SMA, β-actin, and Vimentin were obtained from Affinity Biosciences (Changzhou, China).
5
Cryptolepine Hydrate Modulates Mitochondrial Dynamics
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Purified cryptolepine hydrate (≥98% purity by HPLC), MTT dye, 2′,7′-dichlorofluorescin diacetate, and Rhodamine 123 were purchased from Sigma-Aldrich (St. Louis, MO). The MitoBiogenesis In-cell ELISA kit was purchased from Abcam (Cambridge, MA). Antibodies against c-Myc, p-Drp1, LKB1, and AMPKα1/2 were purchased from Cell Signaling Technology (Beverly MA). The Opa1 antibody was obtained from Thermo Scientific (Rockford, IL). Antibodies against PGC-1α, Mitofusin 1, Mitofusin 2, Drp1, p-AMPKα1/2, SIRT1, and β-actin and horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The ATP Determination kit, MitoTracker Red CMXRos, AlexaFluore 488- and 594-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR).
6
Immunoblot analysis of signaling proteins
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Equal amounts of whole cell lysates were separated on a 10–12% SDS-polyacrylamide gel. The blotting membranes were probed using the following antibodies: NDRG1 (42–6200; Invitrogen), SAPK/JNK (56G8, #9258, Cell Signaling, Danvers, MS, USA), phospho-SAPK/JNK (Thr183/Tyr185, 81E11, #8690, Cell Signaling), p44/42 MAPK (ERK 1/2; 137F5, #4695, Cell Signaling), phospho-p44/42 MAPK (ERK 1/2, Thr202/Tyr204, #9101, Cell Signaling), p38 MAPK (D13E1, #8690, Cell Signaling), phospho-p38 MAPK (Thr180/Tyr182, #9211, Cell Signaling), AMPKα1/2 (#5831, Cell signaling), phospho-AMPKα1/2 (Thr 172; #2535, Cell Signaling), GDF15 (Ab206414, Abcam, Cambridge, MA, USA), alpha-smooth muscle actin (α-SMA) (Ab5694, Abcam), uroplakin II (UPK2) (LS-C41569; LifeSpan BioSciences, Seattle, WA, USA), and β-actin (MAB1501, Merck Millipore, Burlington, MA, USA). Band intensities were detected by the Western lightning plus-ECL detection system (PerkinElmer Inc, Waltham, MA, USA), recorded using the LuminoGraph II (Atto Corporation, Tokyo, Japan), and analyzed using the GeneTool Program of the ChemiGenius (Syngene Cambridge, UK).
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