Rabbit anti ampkα

Manufactured by Cell Signaling Technology

Sourced in United States

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

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22 protocols using rabbit anti ampkα

Effects of SZ-A, FAG, and DAB on Islets and MIN6 Cells

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The islets of KKA^y mice were incubated in RPMI-1640 medium containing vehicle or 100 μg/ml SZ-A for 24 h. MIN6 cells were fasted for 1 h in Krebs buffer (2.8 mM glucose) with or without different concentrations of SZ-A, FAG, or DAB. Then, they were cultured for another 1 h in new Krebs buffer containing 2.8 mM or 16.8 mM glucose combined with SZ-A, FAG, DAB or vehicle. High glucose- and palmitic acid-treated MIN6 cells were coincubated with different concentrations of SZ-A, DNJ or vehicle for 24 h.
Islets or MIN6 cells were homogenized in RIPA lysis buffer (C1053; APPLYGEN, China) containing protease inhibitors (P1265; APPLYGEN, China) and phosphatase inhibitors (P1260; APPLYGEN, China). Protein (10 μg) from each sample was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes for immunoblotting. Rabbit anti-phospho-AMPKα (1:1000, 2535s), rabbit anti-AMPKα (1:1000, 2532s), rabbit-phospho-Erk (1:1000, 9101s), rabbit anti-Erk (1:1000, 9102s), and rabbit anti-HSP90 (1:1000, 4877s) were purchased from Cell Signaling Technology (USA). Goat anti-rabbit IgG/HRP (1:5000, ZDR-5306) was purchased from ZSGB-BIO (China).

Lei L., Huan Y., Liu Q., Li C., Cao H., Ji W., Gao X., Fu Y., Li P., Zhang R., Abliz Z., Liu Y., Liu S, & Shen Z. (2022). Morus alba L. (Sangzhi) Alkaloids Promote Insulin Secretion, Restore Diabetic β-Cell Function by Preventing Dedifferentiation and Apoptosis. Frontiers in Pharmacology, 13, 841981.

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Western Blot Analysis of Phospho-AMPKα

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Samples from the retina or the cell line were isolated and placed in lysis buffer, including protease inhibitor cocktail (Complete, EDTA-free; Roche, Mannheim, Germany) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich) to prepare the lysate, and immunoblot analyses were performed as described elsewhere [2 (link)]. Briefly, the proteins were separated by electrophoresis and electrically transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore; Bedford, MA, USA). The membrane was blocked with 5 % skim milk in Tris-buffered saline with Tween or TNB blocking buffer [0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.5 % TSA Blocking Reagent (PerkinElmer Life Sciences; Waltham, MA, USA)], and then incubated overnight at 4 °C with a rabbit anti-phospho-AMPKα (1:1000; Cell Signaling Technology) or rabbit anti-AMPKα (1:200; Cell Signaling Technology) for normalization. Six samples for each group were analyzed.

Kamoshita M., Fujinami K., Toda E., Tsubota K, & Ozawa Y. (2016). Neuroprotective effect of activated 5′-adenosine monophosphate-activated protein kinase on cone system function during retinal inflammation. BMC Neuroscience, 17, 32.

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Western Blot Analysis of AMPK and Related Signaling

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The cells were harvested in cell lysis buffer (1% sodium dodecyl sulfate, 10 mM TrisHCl, pH 7.5) with a protease inhibitor cocktail (Roche Diagnostics). The measurement of protein concentrations, preparation of samples, and immunoblotting analysis were performed as described.11 Rabbit anti‐AMPKα, rabbit anti‐phospho AMPKα (Thr172), rabbit anti‐acetyl‐coenzyme A carboxylase 1 (ACC1), rabbit anti‐phospho ACC (Ser79), rabbit anti‐microtubule affinity regulating kinase 3 (MARK3), and rabbit anti‐MARK4 antibodies were purchased from Cell Signaling Technology (Danvers, MA). Rabbit anti‐RXRα, mouse anti‐salt inducible kinase 2 (SIK2), and mouse anti‐MARK2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti‐β‐actin antibody (Sigma‐Aldrich) was used as a control for the amount of protein loaded per lane.

Satoh S., Mori K., Onomura D., Ueda Y., Dansako H., Honda M., Kaneko S., Ikeda M, & Kato N. (2017). Ribavirin suppresses hepatic lipogenesis through inosine monophosphate dehydrogenase inhibition: Involvement of adenosine monophosphate‐activated protein kinase‐related kinases and retinoid X receptor α. Hepatology Communications, 1(6), 550-563.

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Protein Expression Analysis in Transfected Cells

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Transfected cells or grinded tumor tissues were lysed in modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% vol/vol NP-40, 1% n-Dodecyl β-D-maltoside, 0.25% wt/vol sodium deoxycholate, 1 mM DTT, and 1 × Roche complete Protease Inhibitor Cocktail) for 1 h at 4 ℃. The lysate was clarified by centrifugation for 20 min at 14,000 × g. The protein concentration was determined using a bicinchoninic acid assay and equal amounts of total protein from each of the samples was supplemented with 5 × SDS loading buffer, incubated at 95 ℃ for 5 min, subjected to SDS-PAGE, followed by western blot analysis. The following antibodies were used: rabbit anti-β-actin (Affinity; 1:5000), rabbit anti- AMPKα (Cell Signaling Technology; 1:1000), rabbit anti-Phospho-AMPKα (Cell Signaling Technology; 1:1000), mouse anti-E-cadherin (Cell Signaling Technology; 1:1000), rabbit anti-N-cadherin (Elabscience; 1:1000), rabbit anti-MMP3 (Proteintech; 1:1000), rabbit anti-GLI1 (Cell Signaling Technology; 1:1000), rabbit anti-GLI2 (NOVAS; 1:1000), goat anti-GLI3 (R&D; 1:1000), mouse anti-IκBα (Cell Signaling Technology; 1:1000), rabbit anti-NF-κB p65 (Cell Signaling Technology; 1:1000) and rabbit anti-Phospho-NF-κB p65 (Cell Signaling Technology; 1:1000).

Cai J., Wang Y., Wang X., Ai Z., Li T., Pu X., Yang X., Yao Y., He J., Cheng S.Y., Yu T., Liu C, & Yue S. (2023). AMPK attenuates SHH subgroup medulloblastoma growth and metastasis by inhibiting NF-κB activation. Cell & Bioscience, 13, 15.

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Placental Protein Expression Analysis

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Phosphorylated and total AMPKα, FABPpm, CD36 and FATP4 protein levels were determined in placental protein extracts by western blot analysis. Plasma membrane and cytosolic protein was isolated from ~50 mg of placental villous tissue from the primary lobe following manufacturer’s directions (Abcam, Plasma Membrane Protein Extraction Kit, ab65400). Protein concentration was quantified by BCA assay. Equal amounts of total protein/sample (20 μg) were separated by SDS-PAGE on a 7.5% gel (TGX^™ Precast Gels, BioRad) and transferred to 0.2 μm nitrocellulose membrane (BioRad). Membranes were blocked with 5% milk TBS-T (TBS+0.01% Tween) buffer for 1 h. Membranes were incubated with primary antibodies rabbit anti-pAMPKα (Thr172) (1:1000, Cell Signaling #9957), rabbit anti-AMPKα (1:1000, Cell Signaling), mouse anti-FABPpm (1:1000 Abcam #ab93928), mouse anti-FATP4 (1:1000 Abnova #H00010999-M01) and rabbit anti-CD36 (1:2000 ThermoFisher #PA1-16813) overnight at 4°C. Membranes were washed in TBS-T before exposure to the appropriate secondary antibody (1:3000, Santa Cruz) for 1 h at room temperature. Antibody binding was detected using a chemiluminescence system (Amersham ECL^™ Prime, GE Healthcare); protein expression was quantified from a digitized image of the blot and expressed as a ratio of phosphorylated to total protein or protein of interest to ponceau S staining.

O’Tierney-Ginn P., Roberts V., Gillingham M., Walker J., Glazebrook P.A., Thornburg K.L., Grove K, & Frias A.E. (2015). Influence of high fat diet and resveratrol supplementation on placental fatty acid uptake in the Japanese macaque. Placenta, 36(8), 903-910.

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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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Western Blot Analysis of Protein Markers

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30–60μg of protein were separated on precast tris-glycine gels (Invitrogen) and transferred to PVDF membrane. Membranes were blocked with 5% BSA/TBST. Primary antibodies, after incubation in 5% BSA/TBST, were detected using HRP conjugated secondary antibodies in chemiluminescent solution using the Quantity One imaging software on a Bio-Rad Gel Docking system. Primary antibodies: Rabbit mAb Bnip3 (EPR4034) from Abcam, rabbit anti-P-AktSer473, rabbit mAb P-Akt308 (C31E5E), rabbit mAb Akt (pan) (C67E7), mouse mAb Histone H3 (96C10), rabbit mAb P-Ser555 ULK (D1H4, #5869), rabbit mAb ULK (D8H5, #8054), rabbit mAb P-AMPKα (Thr172) (40H9, #2535), and rabbit anti-AMPKα (#2532) from Cell Signaling Technology, rabbit anti-LC3B (NB100–2200) from Novus Biologicals, mouse mAb GAPDH from Millipore, rat anti-integrin α6 (GoH3) and mouse anti-HIF1α from BD Pharmingen, mouse mAb AR (441) from Santa Cruz, mouse anti-α tubulin and β-actin-HRP mouse mAb from Sigma-Aldrich), and rabbit anti-integrin α6 (AA6A, A6NT).^{12 (link), 35 (link)}

Nollet E.A., Cardo-Vila M., Ganguly S.S., Tran J.D., Schulz V.V., Cress A., Corey E, & Miranti C.K. (2020). Androgen Receptor-Induced Integrin α6β1 and Bnip3 Promotes Survival and Resistance to PI3K Inhibitors in Castration-Resistant Prostate Cancer. Oncogene, 39(31), 5390-5404.

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Myocardial Protein Analysis by Western Blot

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Total protein was obtained from myocardial tissue and H9C2 cells using pre-cold RIPA lysis buffer and 1% protein phosphatase inhibitor and protein concentration was measured by bicinchoninic acid (BCA) assay. Subsequently, protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After that, PVDF membranes were blocked in 5% milk, incubated with primary antibodies at 4°C overnight, incubated with secondary antibody for 1 h at room temperature and exposed to the ECL in gel imager. Image-Lab software was used to analyze data. The antibodies were used as follows: rabbit anti-AMPKα (5,831, Cell Signaling Technology, Germany), rabbit anti-AMPK-phosphor-T172 (CY5608, Abways, China), rabbit anti-SPTLC1 (DF12752, Affinity, China), rabbit anti-SPTLC2 (DF12231, Affinity, China), mouse anti-GPAT (sc-398135, Santa cruz, China), rabbit anti-CPT-1A (DF12004, Affinity, China), rabbit anti-phosphor-AKT (CY6569, Abways, China), rabbit anti-AKT (CY5551, Abways, China), mouse anti-Bcl-2 (AB3359, Abways, China), rabbit anti-Bax (00089105, proteinch, China), rabbit anti-GAPDH (AB0037, Abways, China).

Tian X., Chen X., Jiang Q., Sun Q., Liu T., Hong Y., Zhang Y., Jiang Y., Shao M., Yang R., Li C., Wang Q, & Wang Y. (2022). Notoginsenoside R1 Ameliorates Cardiac Lipotoxicity Through AMPK Signaling Pathway. Frontiers in Pharmacology, 13, 864326.

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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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Protein Expression Profiling of Cell Lines

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HMEC-1, Vero CCL-81, and HPMEC were sonicated in CelLytic M (Sigma-Aldrich, C2978) supplemented with protease inhibitor cocktail (Sigma-Aldrich, P8340). Following centrifugation at 16000xg for 10 minutes at 4°C, lysates were collected and total protein concentration was determined using DC™ assay (Bio-Rad Laboratories, 5000112).
Equal amounts of proteins (30 μg) were separated on 12% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes (Bio-Rad Laboratories). After blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) supplemented with 0.1% Tween-20, membranes were incubated overnight at 4°C with the following antibodies: rabbit anti-ACE2 (1:1000; abcam, ab272500), rabbit anti-phospho AMPKα Thr172 (1:1000; Cell signaling, 2531), and rabbit anti-AMPKα (1:1000; Cell signaling, 2532). Mouse anti-GAPDH (1:5000; Origene Technologies, TA802519) was used as sample-loading control. The signals were visualised on an Odyssey^®FC Imaging System (LiCor) by infrared (IR) fluorescence using a secondary goat anti-rabbit IRDye 680LT antibody (1:1000; LiCor, FE3680210) and a goat anti-mouse IRDye 800CW (1:1000; LiCor, FE30926210). Bands were quantified through densitometry using the Image Studio Lite 5.0 (LiCor) software.

Perico L., Morigi M., Galbusera M., Pezzotta A., Gastoldi S., Imberti B., Perna A., Ruggenenti P., Donadelli R., Benigni A, & Remuzzi G. (2022). SARS-CoV-2 Spike Protein 1 Activates Microvascular Endothelial Cells and Complement System Leading to Platelet Aggregation. Frontiers in Immunology, 13, 827146.

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