Rabbit anti p ampkα

Manufactured by Cell Signaling Technology

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Rabbit anti-p-AMPKα is a primary antibody that specifically recognizes the phosphorylated form of the AMP-activated protein kinase (AMPK) alpha subunit. AMPK is a key cellular energy sensor that plays a crucial role in regulating cellular metabolism and energy homeostasis.

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AMPKα (280 citations) P-AMPKα (127 citations) Anti-AMPKα (120 citations) Anti-p-AMPKα (25 citations) Rabbit anti-AMPKα (22 citations)

7 protocols using rabbit anti p ampkα

Western Blot Analysis of AMPK and Adiponectin

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Adipose tissue and serum-starved J774 macrophages were homogenised in RIPA lysis buffer. Briefly, 40 μg protein was loaded onto a 16% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes (0.2 μm pore size) [31 (link)]. Proteins were identified using rabbit anti-p-AMPKα (Thr172; no. 2535s), rabbit anti-AMPKα (no. 2532s) and rabbit anti-adiponectin (no. 2789) antibodies (all diluted 1:1000; Cell Signaling, Danvers, MA, USA). Proteins were normalised against β-actin (mouse anti-β-actin antibody [no. A2228]; Sigma, St Louis, MO, USA), diluted 1:10,000 (see ESM Methods for further details). Original western blot images were cropped as indicated by vertical lines.

Börgeson E., Wallenius V., Syed G.H., Darshi M., Lantero Rodriguez J., Biörserud C., Ragnmark Ek M., Björklund P., Quiding-Järbrink M., Fändriks L., Godson C, & Sharma K. (2017). AICAR ameliorates high-fat diet-associated pathophysiology in mouse and ex vivo models, independent of adiponectin. Diabetologia, 60(4), 729-739.

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Crocetin Modulates TGF-β Signaling

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Crocetin was obtained from MP Biomedicals, CA, United States. Crocetin was purified by high-performance liquid chromatography (HPLC) with 98% purity and then dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) with a 0.1% final concentration in the culture medium. TGF-β was purchased from Merck Millipore (Burlington, MA, United States). Antibodies including rabbit anti-Caspase3 (#2723), mouse anti-Bcl-2 (#15071), mouse anti-Bax (#89477), rabbit anti-TGF-β (#3711), rabbit anti-α-SMA (#19245), rabbit anti-p-AMPKα (#50081), rabbit anti-p-Beclin-1 (#35955), mouse anti-SIRT-1 (#8469), rabbit anti-LC3 (#12741), and rabbit anti-NDRG2 (#5667) were purchased from Cell Signaling Technology (CST) Inc., Beverly, MA, United States. Mouse anti-GAPDH (sc-365062) and mouse anti-p-mTOR (sc-293113) were obtained from Santa Cruz Biotechnology, Inc., CA, United States.

Guo H., Ruan C., Zhan X., Pan H., Luo Y, & Gao K. (2022). Crocetin Protected Human Hepatocyte LO2 Cell From TGF-β-Induced Oxygen Stress and Apoptosis but Promoted Proliferation and Autophagy via AMPK/m-TOR Pathway. Frontiers in Public Health, 10, 909125.

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AMPK Signaling Pathway Characterization

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OFs were lysed and processed as described previously.²⁰ (link) Antibodies targeting p-AMPKα (rabbit anti-p-AMPKα), AMPKα (rabbit anti-AMPKα), p-AMPKβ (rabbit anti-p-AMPKβ), AMPKβ1/2 (rabbit anti-AMPKβ1/2), p-ACC (rabbit anti-p-ACC), ACC (rabbit anti-ACC), and β-tubulin (rabbit anti-β-tubulin) were all obtained from Cell Signaling Technology (Danvers, MA, USA), and AMPKβ (mouse anti-AMPKβ) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Hammond C.L., Roztocil E., Gonzalez M.O., Feldon S.E, & Woeller C.F. (2021). MicroRNA-130a Is Elevated in Thyroid Eye Disease and Increases Lipid Accumulation in Fibroblasts Through the Suppression of AMPK. Investigative Ophthalmology & Visual Science, 62(1), 29.

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Intracellular Signaling Pathway Analysis

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Media and reagents were acquired from the following sources: DMEM and Trypsin from Corning; FBS from Gibco; cell lysis buffer from Cell Signaling Technology; angiotensin II, WP1066, AICAR and compound C from EMD Millipore; (GFP)-fused LC3B (pEGFP-LC3) from Addgene; and ADP/ATP assay kit from Sigma. Antibodies were acquired from the following sources: rabbit anti-p-STAT3 (Tyr705), rabbit anti-p-STAT3 (Ser727), rabbit anti-STAT3, rabbit anti-p-mTOR (Ser2448), anti-mTOR, rabbit anti-p-AMPKα (T172), rabbit anti-AMPKα, rabbit anti-p-JAK2 (Tyr1007/1008) and rabbit anti-p-4E-BP1 (T37/46) from Cell Signaling; rabbit anti-LC3Bfrom Novus Biologicals; rabbit anti-β-Actin and rabbit anti-p62 from Santa Cruz Biotechnology.

Chen L., Zhao L., Samanta A., Mahmoudi S.M., Buehler T., Cantilena A., Vincent R.J., Girgis M., Breeden J., Asante S., Xuan Y.T, & Dawn B. (2017). STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis. PLoS ONE, 12(7), e0179835.

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Comprehensive Antibody Characterization for Western Blotting and Immunostaining

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Primary antibodies used for Western blotting and immunostaining were chicken anti-HMGCS2 (Genway, San Diego, CA), rabbit anti-HMGCS2 (AVIVA, San Diego, CA; epitope different from the Genway antibody), chicken anti-MCT1 (Chemicon, Temecula, CA), rabbit anti-MCT1, goat anti-PPARα (Santa Cruz Biotechnology, Dallas, TX), rabbit anti–α-smooth muscle actin, rabbit anti-prohibitin (Abcam, Cambridge, UK), rabbit anti–cytochrome c, rabbit anti-Glut4, rabbit anti–COX IV, rabbit anti-ACC, rabbit anti–phospho-ACC, rabbit anti-AMPKα, rabbit anti–P-AMPKα, rabbit anti-AMPKβ1, rabbit anti–P-AMPKβ1 (Cell Signaling Technology, Danvers, MA), rat anti-Hsc70 (Enzo Life Sciences, Helsinki, Finland), rat anti-K8 (Troma I Developmental Studies Hybridoma Bank, Iowa City, IA), and mouse anti-tubulin (Sigma-Aldrich, Munich, Germany). Secondary antibodies used for Western blotting were anti-mouse immunoglobulin G (IgG)–horseradish peroxidase (HRP), anti-rat IgG-HRP (GE Healthcare, Buckinghamshire, UK), anti-rabbit IgG-HRP (Cell Signaling Technology), anti-chicken IgG-HRP (Genway), and anti-goat IgG-HRP (Cell Signaling Technology). Secondary antibodies used for immunostaining were anti-rabbit Alexa Fluor 488 and anti-rat Alexa Fluor 546 (Life Technologies, Carlsbad, CA). Nuclei were stained with Toto-3 (Life Technologies) or DRAQ5 (Cell Signaling Technology).

Helenius T.O., Misiorek J.O., Nyström J.H., Fortelius L.E., Habtezion A., Liao J., Asghar M.N., Zhang H., Azhar S., Omary M.B, & Toivola D.M. (2015). Keratin 8 absence down-regulates colonocyte HMGCS2 and modulates colonic ketogenesis and energy metabolism. Molecular Biology of the Cell, 26(12), 2298-2310.

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Western Blot Analysis of AMPK Activation

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Whole proteins of treated HepG2 cells were extracted using RIPA buffer with 1% protease inhibitor cocktail. Lysates at 10 μg concentration were added to an SDS-PAGE gel and subjected to electrophoresis, and transferred onto nitrocellulose membranes (Millipore, USA). The primary antibodies, including rabbit anti-pAMPKα (#2531, Cell Signaling Technology, MA, USA), rabbit anti-AMPKα (#2532, Cell Signaling Technology, MA, USA) and mouse anti-β-actin (ab8226, Abcam, USA) were used. The corresponding HRP-conjugated secondary antibodies (Abcam, USA) were used. Expression of targeted proteins were visualized by TMB chemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression level of targeted proteins was normalized to β-actin expression compared with the control group.

Sangpairoj K., Pranweerapaiboon K., Saengkhae C., Meemon K., Niamnont N., Tamtin M., Sobhon P., Yisarakun W, & Siangcham T. (2024). Extracts of tropical green seaweed Caulerpa lentillifera reduce hepatic lipid accumulation by modulating lipid metabolism molecules in HepG2 cells. Heliyon, 10(6), e27635.

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Western Blot Analysis of Fly Head Proteins

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Fly heads were homogenized in Laemmli sample buffer (#1610737, Bio-Rad, Hercules, CA). For separation of polypeptides, samples were electrophoresed through precast polyacrylamide gels (Bio-Rad, #4561094) for 1 hour in a Tris/glycine/SDS buffer. Separated proteins were transferred to a nitrocellulose membrane and were then blocked with Odyssey blocking buffer (LI-COR Lincoln, NE, #927–40000) for 1 hour prior to incubation overnight with rabbit anti-pAMPKα (1:1000, Cell Signaling technology, Danvers, MA, #2535) and mouse anti-α-Tubulin (Sigma #T9026, diluted 1:5000) antibody at 4 °C. After 3 x 15-minute washes in PBS at RT, samples were incubated with IRDye 680RD and 800CW secondary antibodies diluted 1:10,000 (LI-COR) for 30 minutes at RT. Western blots were imaged using an Odyssey Fc imaging system (LI-COR) and ImageJ was used to quantify probe signal intensity.

Nagy S., Maurer G.W., Hentze J.L., Rose M., Werge T.M, & Rewitz K. (2018). AMPK signaling linked to the schizophrenia-associated 1q21.1 deletion is required for neuronal and sleep maintenance. PLoS Genetics, 14(12), e1007623.

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