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Anti phospho ampkα

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Anti-phospho-AMPKα is a laboratory reagent used to detect and quantify the phosphorylation of the AMP-activated protein kinase (AMPK) α subunit. AMPK is a critical cellular energy sensor that plays a key role in regulating metabolism. The anti-phospho-AMPKα antibody specifically recognizes the phosphorylated form of the AMPK α subunit, allowing researchers to study AMPK activation in response to various cellular stimuli.

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Lab products found in correlation

Anti ampkα, by Cell Signaling Technology (24 mentions) Anti phospho akt, by Cell Signaling Technology (7 mentions) Anti phospho mtor, by Cell Signaling Technology (6 mentions) Anti gapdh, by Cell Signaling Technology (5 mentions) Anti phospho yap, by Cell Signaling Technology (5 mentions) Anti akt, by Cell Signaling Technology (5 mentions) Anti mtor, by Cell Signaling Technology (5 mentions) Anti yap, by Cell Signaling Technology (4 mentions) Anti β actin, by Cell Signaling Technology (4 mentions) Anti p70 s6 kinase, by Cell Signaling Technology (4 mentions) Anti phospho acetyl coa carboxylase, by Cell Signaling Technology (4 mentions) Anti β actin, by Merck Group (4 mentions) Anti phospho erk, by Cell Signaling Technology (3 mentions) Anti phospho acc, by Cell Signaling Technology (3 mentions) Anti phospho lats1, by Cell Signaling Technology (3 mentions) Anti phospho lkb1, by Cell Signaling Technology (3 mentions) Anti caspase 3, by Cell Signaling Technology (3 mentions) Anti phospho p70s6k, by Cell Signaling Technology (3 mentions) Alexa fluor 488 labeled donkey anti rabbit igg antibody, by Thermo Fisher Scientific (3 mentions) Eclipse 80i, by Nikon (3 mentions)

Close spelling variants of this product

Anti-phospho-AMPKα (Thr172) Anti-phospho-T172 AMPKα ( Anti-phospho-AMPKα monoclonal antibody 40H9 Anti-phospho-AMPKα (Thr172) antibody Anti-phospho-AMPKα (Thr172) (40H9; Rabbit anti-phospho-AMPKα Rabbit anti-phospho-AMPKα T172 Rabbit anti-phospho-AMPKα (Thr172) (2535 Rabbit anti-phospho-AMPKα (Thr172) Phospho-AMPKα

48 protocols using anti phospho ampkα

1

Cordycepin Modulates AMPK and mTOR Pathways

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Cordycepin specified to be over 99.2% pure as determined by HPLC was purchased from Shanghai Winherb Medical Co. (Shanghai, China). Primary antibodies against the following proteins were obtained from Cell Signalling Technology (Danvers, MA): anti‐AMPKα (#2603P), anti‐phospho‐AMPKα (#2535), anti‐mammalian target of rapamycin (mTOR) (#2983), anti‐phospho‐ mTOR (#2971), anti‐phospho‐ERK, #4370P), anti‐ERK (#4695), anti‐acetyl‐CoA carboxylase (ACC) (#3676), anti‐phospho‐ACC (#3661), and anti‐GAPDH (#2118). Primary antibodies against gp91phox (ab129068), superoxide dismutase 1 (SOD1, ab16831), SOD2 (ab38155), and α‐actinin (ab68167) were purchased from Abcam (Cambridge, UK). The secondary antibody was purchased from LI‐COR Biosciences. Ang II (A9525) and compound C (CpC, P5499) were purchased from Sigma‐Aldrich. Proteins were measured with assay kits obtained from Pierce (Pierce, 23225).
Wang H., Duan M., Xu M., Huang S., Yang J., Yang J., Liu L., Huang R., Wan C., Ma Z., Wu Q, & Tang Q. (2019). Cordycepin ameliorates cardiac hypertrophy via activating the AMPKα pathway. Journal of Cellular and Molecular Medicine, 23(8), 5715-5727.
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2

Phospho-AMPK Immunoblotting in CD45-CD31- Cells

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CD45–CD31– cells isolated from LNs as described above were immediately lysed on ice in lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 2 mM NaF and 0.01% NP40) containing 1 mM sodium orthovanadate (phosphatase inhibitor), 50 mM PMSF, and protease inhibitor cocktail (Calbiochem). Samples were boiled in 4× sample buffer (Bio-Rad) and immunoblotted using anti-Phospho-AMPKα and anti-AMPKα (Cell Signaling Technology).
Majumder S., Amatya N., Revu S., Jawale C.V., Wu D., Rittenhouse N., Menk A., Kupul S., Du F., Raphael I., Bhattacharjee A., Siebenlist U., Hand T.W., Delgoffe G.M., Poholek A.C., Gaffen S.L., Biswas P.S, & McGeachy M.J. (2019). IL-17 metabolically reprograms activated fibroblastic reticular cells for proliferation and survival. Nature immunology, 20(5), 534-545.
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3

Molecular Mechanisms of CRAC Channel Regulation

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All plasmids used in this study were sequenced to confirm their identity. Crbn, Ampk, Slo, or HA-Ubiquitin constructs used in this study were described previously26 (link). Orai1 (MMM1013-202764440), Orai2 (MMM1013-202859855), and Orai3 (MMM1013-202842392) cDNA were purchased from Open Biosystems to construct expression vectors by a PCR-based strategy. Glutamine synthetase (GS) cDNA were synthesized from mRNA of the testis of mice and GS expression vectors were generated. The complete list of all primer sequences used in the study is provided as a Supplementary table. Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.
Moon H., Min C., Kim G., Kim D., Kim K., Lee S.A., Moon B., Yang S., Lee J., Yang S.J., Cho S.K., Lee G., Lee C.S., Park C.S, & Park D. (2020). Crbn modulates calcium influx by regulating Orai1 during efferocytosis. Nature Communications, 11, 5489.
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4

Protein Extraction and Western Blot Analysis

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The protein extraction method was according to our previous study19 ,22 (link). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the separated proteins were transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 1% casein at room temperature for 1 h. Subsequently, the membrane incubated overnight at 4 °C with the primary antibodies against antiphospho-AMPKα (1: 1000, #2535, Cell Signaling Technology), antiAMPKα (1: 1000, #5831, Cell Signaling Technology), and antiβ-actin (1:3000, A1978, Millipore Sigma). After washing with TBST for four times, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using Tanon 4200SF system (Tanon Biotechnology, Shanghai, China) and quantified densitometry using Image J software (U.S. National Institutes of Health, Bethesda, MD).
Wang S.W., Sheng H., Bai Y.F., Weng Y.Y., Fan X.Y., Lou L.J, & Zhang F. (2020). Neohesperidin enhances PGC-1α-mediated mitochondrial biogenesis and alleviates hepatic steatosis in high fat diet fed mice. Nutrition & Diabetes, 10, 27.
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5

Molecular Mechanisms of SC Herb in Metabolic Regulation

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Anti-SREBP-1c, anti-phospho-AMPKα, anti-phospho-acetyl-CoA carboxylase (ACC), anti-phospho- liver kinase B1 (LKB1), anti-phospho-YAP, anti-YAP, anti-phospho-large tumor suppressor kinase 1 (LATS1), anti-heme oxygenase 1 (HO-1), anti-Lamin A/C, and anti-β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Nrf2 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Trizol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Compound C (Com C), verteporfin (VP), T0901317 (T090), Harris hematoxylin and eosin, and Oil Red O solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol (PEG) 400 was obtained from Yakury Pure Chemical Co., Ltd. (Kyoto, Japan). SC is a medicinal-standard herb manufactured at a GMP facility (Nonglim Saengyak, Seoul, Republic of Korea) certified by the Korean FDA and was prepared and standardized as previously described [13 (link)].
Bae S.J., Lee W.Y., Bak S.B., Kim Y.E., Kim M.J, & Kim Y.W. (2023). Unraveling the Antioxidant Capacity of Spatholobi caulis in Nonalcoholic Fatty Liver Disease: A Multiscale Network Approach Integrated with Experimental Validation. Antioxidants, 12(5), 1097.
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6

Immunoblotting Protocol for Protein Quantification

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Immunoblotting was performed as described in our previous reports (11 (link)). In brief, cells were lysed and total protein was quantified using the Bradford assay (Bio-Rad Laboratories Lnc.). An equal amount of total protein (20–25 μg) in each sample was loaded onto an SDS-PAGE gel. After transferring to PVDF membrane, the blot was incubated with antibodies. Antibodies were diluted as follows: anti-Cx43 (Sigma-Aldrich Co., 1:4000), anti-MGMT (Cell Signaling Technology®, 1:300), anti-β-actin (Sigma-Aldrich Co. LLC, 1:10000), anti-phospho-AKT (Cell Signaling Technology®, 1:1000), anti-AKT (Cell Signaling Technology®, 1:1000), anti-phospho-AMPKα (Cell Signaling Technology®, 1:1000), anti-AMPKα (Cell Signaling Technology®, 1:1000), anti-phospho-MTOR (Cell Signaling Technology®, 1:1000), anti-MTOR (Cell Signaling Technology®, 1:1000), anti-GFAP (Cell Signaling Technology®, 1:500), anti-NOTCH1 (Bio-Rad Laboratories Inc., 1:1000), and anti-GAPDH (Santa Cruz Biotechnology, Inc., 1:5000). Images were taken using a ChemiDoc™ MP System (Bio-Rad Laboratories Lnc). The intensity of the band was quantified using Image Lab software (Bio-Rad Laboratories Lnc.) or ImageJ as described earlier(11 (link)). The relative level of Cx43 proteins (Cx43/ACTB) is defined as the ratio of band intensity of Cx43 to that of β-actin.
Murphy S.F., Varghese R.T., Lamouille S., Guo S., Pridham K.J., Kanabur P., Osimani A.M., Sharma S., Jourdan J., Rodgers C.M., Simonds G.R., Gourdie R.G, & Sheng Z. (2015). Connexin 43 inhibition sensitizes chemoresistant glioblastoma cells to temozolomide. Cancer research, 76(1), 139-149.
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7

Western Blot Analysis of Autophagy Proteins

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Cells were lysed with RIPA buffer (#P0013C, Beyotime, China). Protein concentration was quantified using a BCA protein assay kit (#P0012S, Beyotime, China) and transferred to PVDF membrane (# 88518, Thermo Fisher Scientific, USA) after separation on SDS-PAGE gels. The membranes were incubated with primary antibodies at 4°C for 12 h, and then washed three times with TBST buffer. Finally, the membranes were incubated with HRP-conjugated secondary antibodies for observation. An image processing system (Bio-Rad) and ImageJ software were used to determine and quantify these protein bands, respectively. Primary antibodies and a secondary antibody, including anti-GBP1 (#PA5– 23509), were purchased from Thermo Fisher Scientific. The other antibodies, including anti-GAPDH (#5174), anti-LC3A/B (#12741), anti-Beclin-1 (#3738), anti-AMPKa (#5831), anti-phospho-AMPKα (#50081), anti-mTOR (#2983), anti-phospho-mTOR (#2971), anti-ULK1(#8054), anti-phospho-ULK1 (#6887) and anti-rabbit IgG-HRP-linked antibody (#7074) were purchased from Cell Signaling Technology. The ratio of target protein/GAPDH or LC3II/LC3I is shown below the protein band.
Yu Y., Pan J., Liu M., Jiang H., Xiong J., Tao L., Xue F., Tang F., Wang H, & Dai J. (2022). Guanylate-Binding protein 2b regulates the AMPK/mTOR/ULK1 signalling pathway to induce autophagy during Mycobacterium bovis infection. Virulence, 13(1), 875-889.
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8

Quantifying Hepatic CSE and CBS Levels

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To confirm the CSE and CBS protein expression levels, 10 μg protein samples lysed in RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Thermo Scientific) were prepared from the livers taken from each group. The proteins were resolved on a 10% gel and transferred to a polyvinylidine difluoride membrane (Millipore, Bedford, MA, USA). The membrane was then incubated overnight with primary antibodies at 4 °C. Anti-CSE (MBS2015730) was obtained from My BioSource (San Diego, CA, USA), and anti-CBS (#14782), anti-TNF-α (#6945), anti-IL-6 (#12153), anti-phospho-NF-κB p65 (#3033), anti-NF-κB p65 (#4764), anti-phospho-AMPKα (#2535), anti-AMPKα (#2532), and anti-β-actin (#4967) were obtained from Cell Signaling Technology (Canvers, MA, USA). Anti-PEPCK was purchased from Santa Cruz (Dallas, TX, USA). The secondary antibodies were incubated for 1 h at room temperature. Anti-Rabbit (#7074) and anti-Mouse (#7076) were obtained from Cell signaling Technology (Canvers, MA, USA). Bands were detected using an enhanced chemiluminescence system (Amersham, Buckinghamshire, UK).
Na A.Y., Jo J.J., Kwon O.K., Cho P., Gao Y., Kim J.H., Kim K.M., Ki S.H, & Lee S. (2021). Characterization of hyperglycemia due to sub-chronic administration of red ginseng extract via comparative global proteomic analysis. Scientific Reports, 11, 12374.
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9

Investigating PPAR-α Signaling Pathways

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BZA was acquired from Sigma (B7273, purity > 98%). Phenylephrine (PE, P1240000) was obtained from Sigma-Aldrich. Anti-PPAR-α (sc-9000) and anti-PCNA (sc-7907) were purchased from Santa Cruz Biotechnology. The following first antibodies were purchased from Cell Signaling Technology: anti-AKT (#4691), anti-phospho-AKT (#4060), anti-GSK3β (#9315), anti-phospho-GSK3β (#9323P), anti-ERK (#4695), anti-phospho-ERK (#4370P), anti-P38 (#9212P), anti-phospho-P38 (#4511P), anti-AMPKα (#2603P), and anti-phospho-AMPKα (#2535). Anti-GAPDH (#ab8245), anticalcineurin (CaN) (#ab90540), and anti-NFAT1 (#ab2722) were obtained from ABCAM. Anti-α-actinin was acquired from Millipore. The secondary antibodies were purchased from LI-COR Biosciences. The PPAR-α antagonist (GW6471, G5045), PPAR-β/δ antagonist (GSK0660, G5797), and PPAR-γ antagonist (GW9662, M6191) were all purchased from Sigma-Aldrich. All other chemicals were of analytical grade.
Xu S.C., Ma Z.G., Wei W.Y., Yuan Y.P, & Tang Q.Z. (2017). Bezafibrate Attenuates Pressure Overload-Induced Cardiac Hypertrophy and Fibrosis. PPAR Research, 2017, 5789714.
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10

Protein Extraction and Western Blotting

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Total protein was extracted from frozen hearts, resolved and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by standard Western blotting procedures [7] (link). A total of 20 µg protein was fractionated using NuPAGE 4–12% Bis-Tris (Invitrogen) gels and transferred to a Protran nitrocellulose transfer membrane (Whatman). The antibodies used for Western blotting were as follows: anti-CaMKKβ (H-95) (Santa Cruz Biotechnology, Dallas, TX, USA), 1∶500; anti-total-CaMKI (sc-33165) (Santa Cruz Biotechnology), 1∶1000; anti-phospho-CaMKIV (sc-28443-R) (Santa Cruz Biotechnology), 1∶500; anti-total-CaMKIV (sc-55501) (Santa Cruz Biotechnology), 1∶1000; anti-phospho-AMPKα (Cell Signaling Technology, Beverly, MA, USA), 1∶500; anti-total-AMPKα (Cell Signaling Technology), 1∶1000; and anti-GAPDH (Cell Signaling Technology), 1∶2000. Anti-phospho-CaMKI (Thr177) was a kind gift from Dr. Naohito Nozaki (Kanagawa Dental College, Yokosuka, Kanagawa, Japan) and used at the dilution of 1∶50. Anti-rabbit IgG (GE Healthcare) and anti-mouse IgG (GE Healthcare) were used as secondary antibodies at a dilution of 1∶2000.
Watanabe S., Horie T., Nagao K., Kuwabara Y., Baba O., Nishi H., Sowa N., Narazaki M., Matsuda T., Takemura G., Wada H., Hasegawa K., Kimura T, & Ono K. (2014). Cardiac-Specific Inhibition of Kinase Activity in Calcium/Calmodulin-Dependent Protein Kinase Kinase-β Leads to Accelerated Left Ventricular Remodeling and Heart Failure after Transverse Aortic Constriction in Mice. PLoS ONE, 9(9), e108201.
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