The cells were harvested in lysis buffer (50 mM Tris–HCl, pH7.4, 150 mM NaCl, 20 mM sodium pyrophosphate, 10 mM NaF, 2 mM sodium orthovanadate, 1 mM EDTA, 1% Triton X-100, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 6500 IU/ml aprotinin, phosphatase inhibitor mixture-1, Sigma), and after end-over-end rotation of the homogenates for 30 min, lysate supernatants were collected by centrifugation (15,000 rpm) for 20 min at 4°C, and protein concentrations were measured using the bicinchoninic acid method with bovine serum albumin (BSA) as the standard (Pierce). After electrophoresis, western blotting was performed following standard procedures, and chemiluminescence was detected with ImageQuant LAS4000 mini (GE Healthcare). Quantification was performed with ImageQuant TL (GE Healthcare). Antibodies against RSPO3 (ATLAS Antibodies, #HPA029957), phospho-Acetyl-CoA Carboxylase (ACC) (Ser79) (Cell Signaling, #3661), phospho-AMPKα (Thr172) (Cell Signaling, #2535), and β-actin (Sigma, #A2228) were used for Western blot analysis.
Phospho acetyl coa carboxylase ser79
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-Acetyl-CoA Carboxylase (Ser79) is a lab equipment product that detects the phosphorylation of Acetyl-CoA Carboxylase at serine 79. This modification is important for the regulation of Acetyl-CoA Carboxylase activity.
Automatically generated - may contain errors
Lab products found in correlation
Phospho ampkα thr172, by Cell Signaling Technology (5 mentions)
Acetyl coa carboxylase, by Cell Signaling Technology (5 mentions)
β actin, by Merck Group (3 mentions)
Metformin, by Merck Group (2 mentions)
Ampkα, by Cell Signaling Technology (2 mentions)
Ampkα1, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Phospho ampk thr172, by Cell Signaling Technology (2 mentions)
Phosphatase inhibitor mixture 1, by Merck Group (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Palmitoleic acid, by Merck Group (1 mentions)
Bovine insulin, by Merck Group (1 mentions)
Dmem no glucose, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
α linolenic acid, by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
16 protocols using phospho acetyl coa carboxylase ser79
1
Cell Lysis and Protein Quantification
2
Palmitate-Induced Metabolic Dysregulation
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Mouse C2C12 skeletal muscle cells were obtained from the ATCC (Rockville, USA). Fetal bovine serum (FBS), horse serum, Dulbecco's modified eagle medium (DMEM), no-glucose DMEM, Dulbecco's phosphate buffered saline (DPBS), and Antibiotic-Antimycotic were from Gibco (Grand Island, USA). Fatty acid free-bovine serum albumin (BSA) was obtained from Fitzgerald (North Acton, USA). Palmitic acid (PA), palmitoleic acid (POA), oleic acid (OA), linoleic acid (LA), α-linolenic acid (ALA), bovine insulin, metformin, and adenine 9-β-D-arabinofuranoside (araA) were obtained from Sigma-Aldrich (St. Louis, USA). 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) was obtained from Invitrogen (Carlsbad, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Amresco (Solon, USA). The antibodies specific for phospho-Akt (Ser473), Akt, phospho-PKCθ (Thr538), PKCθ, phospho-JNK (Thr183/Tyr185), JNK, phospho-AMPKα (Thr172), AMPKα, phospho-acetyl-CoA carboxylase (ACC) (Ser79), and ACC were obtained from Cell Signaling Technology (Danvers, USA).
3
Western Blot Analysis of Autophagy-Related Proteins
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Cellular lysates were prepared with lysis buffer (Cell Signaling, Beverly, MA, USA) and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10-12% polyacrylamide gels and transferred to polyvinyldifluoride membranes. The membranes were incubated in blocking buffer (TBS/T; 5% non-fat milk in 0.2 M Tris, 1.36 M NaCl, 0.1% Tween 20) for 1 h at room temperature, and then washed three times for 5 min each with 15 mL of TBS/T. Membranes were incubated overnight with the primary antibodies at 4℃. Antibodies against SIRT1, p21, Microtubule-associated protein 1A/1B-light chain 3 (LC3) LC3A/B, p62, phospho-AMPK, total AMPK, and Phospho-Acetyl-CoA Carboxylase (Ser79) were purchased from Cell Signaling. An anti-GAPDH antibody was purchased from Sigma. The membranes were washed three times with TBS/T and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody for 1 h at 25℃. After washing three times with TBS/T, the membranes were incubated with Pierce™ ECL Western Blotting Substrate (Invitrogen) and exposed to film.
4
Western Blot Analysis of Signaling Proteins
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Pelleted cells were homogenized by sonication in lysis buffer [20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 1% Nonidet P-40, 2 mM Na3VO4, protease inhibitor (10 μL per mL) and phosphatase inhibitor (20 μg per mL)], followed by centrifugation at 1200 × g for 10 min at 4 °C. Protein concentrations in supernatants were determined using a bicinchoninic acid (BCA) protein assay kit (23255; Pierce, Thermo Fisher Scientific) and bovine serum albumin as standard. Cell lysates (20 µg protein) were subjected to standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were transferred to a nitrocellulose membrane. Proteins were reversibly visualized using MemCode stain (24580; MemCode Reversible protein Stain, Pierce, Thermo Fisher Scientific) and detected using antibodies against phosphor-AKTSer473 (sc-33437, Santa Cruz Biotechnology), total AKT (05-591, Millipore), phospho-FOXO1Thr24/FOXO3aThr32 (#9464, Cell Signaling), total FOXO1 (#2880, Cell Signaling), phospho-acetyl-CoA carboxylaseSer79(#3661, Cell Signaling), and total acetyl-CoA carboxylase (#3661, Cell Signaling). Densitometric analysis was performed using Image lab 5.0 software from BioRad. Protein expression data were corrected to protein stain.
5
Immunoblotting Analysis of Signaling Pathways
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The procedure was performed as described previuosly 31 . The following antibodies were used: anti-pY (1068) EGFR (#3777), anti-PARP (#9542), anti-pS(235/236)S6 (#4858), anti- S6 (#2217), Phospho-4E-BP1 (Thr37/46) (236B4) (#2855), 4E-BP1 (53H11) (#9644), Phospho-Akt (Ser473) (D9E) XP, (#4060), Acetyl-CoA Carboxylase (C83B10)( #3676), Phospho-Acetyl-CoA Carboxylase (Ser79) (#3661), all were purchased from Cell Signaling Technology (Beverly, MA). Anti-ERK pT(202)Y(204) (E-4), Akt 1/2/3 (H-136), anti-EGFR(A-10), anti-ERK2 (c-14) and GAPDH (FL-335) were from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated goat anti-mouse and HRP-conjugated goat anti-rabbit were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
6
Characterization of Cancer Stem Cell Markers
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ABCG2 (AbCam, ab3380); CD133 (Miltenyl Biotec, W6B3C1); ALDH1A1 (AbCam, ab52492); Acetyl‐CoA Carboxylase (Cell Signaling, 3676); Phospho‐Acetyl‐CoA Carboxylase (Ser79) (Cell Signaling, 3661); AMPK (Cell Signaling, 2532); Phospho‐AMPKα (Thr172)(Cell Signaling, 2531); EZH2 (Bethyl, A304‐197A); GAPDH (Bethyl, A300‐641A); Actin (Santa Cruz, sc‐1616); Complex II (MitoScience, MS204); Complex IV (MitoScience, MS404); Compound C (Calbiochem, 171260); Iodotubercin (Sigma, I100); MitoSox (Invitrogen, M36008); Mitotracker Red (Invitrogen, M7512).
7
Western Blot Analysis of Liver Protein Expression
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The liver samples were homogenized in lysis buffer (containing proteinase and phosphatase inhibitors) using a motor-driven pestle in a 1.5-ml conical tube. After being stored on ice for 30 min, the homogenates were centrifuged at 4°C at 12,000 x g for 45 min. The supernatants were harvested, and the protein concentrations were measured. Western blotting followed the procedure of Murase et al. [11 (link)]. The bands of the target protein were obtained using a chemiluminescence reagent (Thermo Scientific, MA) and a ChemiDoc XRS imaging system (Bio-Rad, CA).
Primary antibodies for ACACA (Thermo, PA5-17564), phospho-acetyl-CoA carboxylase (Ser79) (Cell Signaling Technologies, #03661, MA), phospho-AMPKα (Thr172) (Millipore, #07–626, MA), and GAPDH (Santa cruz, sc-20357, TX) were used in this experiment. These antibodies were all validated for use with chicken samples according to the instructions of the companies.
Primary antibodies for ACACA (Thermo, PA5-17564), phospho-acetyl-CoA carboxylase (Ser79) (Cell Signaling Technologies, #03661, MA), phospho-AMPKα (Thr172) (Millipore, #07–626, MA), and GAPDH (Santa cruz, sc-20357, TX) were used in this experiment. These antibodies were all validated for use with chicken samples according to the instructions of the companies.
8
Metformin and Compound C Mechanism
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Metformin hydrochloride (D150959) was purchased from Sigma-Aldrich (St Louis, MO, USA) and dissolved in sterile saline. Compound C (6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo[1,5-a] pyrimidine) was obtained from Tocris (Cat. No. 3093) and dissolved in dimethyl sulfoxide and further diluted with sterile saline. Metformin and Compound C (10 mg/kg) were injected intraperitoneally. Phospho-Acetyl-CoA Carboxylase (Ser79) (3661, 1:500), Acetyl-CoA Carboxylase (C83B10) rabbit mAb (3676, 1:1000), AMPKα (D63G4) rabbit mAb (5832, 1:1000) and Phospho-AMPKα (Thr172) (40H9) rabbit mAb (2535, 1:1000) were obtained from Cell signaling technology (Beverly, MA, USA). The mouse monoclonal antibody β-actin (A1978, 1:5000) was the product of Sigma-Aldrich (St Louis, MO, USA). HRP-conjugated secondary antibodies for western blot were diluted as 1: 10,000, and were from Thermo Fisher Scientific (Waltham, MA, USA).
9
Analyzing Obesity and Metformin Effects on Endometrial Proliferation
10
Western Blot Analysis of AMPK Pathway
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Whole-cell lysates were resolved by SDS-PAGE gel electrophoresis, electrotransferred to a PVDF membrane, and probed with indicated antibodies. The following antibodies were purchased from Cell Signaling Technology: AMPKα1 (#2795), AMPKα2 (#2757), Phospho-AMPKα (Thr172) (#2535), AMPKβ1 (#4178), AMPKβ2 (#4148), AMPKγ1 (#4187), AMPKγ2, (#2536), AMPKγ3 (#2550), Acetyl-CoA Carboxylase (#3676), Phospho-Acetyl-CoA Carboxylase (Ser79) (#3661), cleaved Caspase-3 (Asp175) (#9661 and #9664), E-cadherin (#3195), Gasdermin D (#97558 and #93709), and cleaved Gasdermin D (Asp275) (#36425).
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