Blocking Assay:Article Title: Chronic Cannabidiol Administration Attenuates Skeletal Muscle De Novo Ceramide Synthesis Pathway and Related Metabolic Effects in a Rat Model of High-Fat Diet-Induced Obesity
Article Snippet: .. After blocking in Tris-buffered saline with Tween-20 (TBST) with 5% non-fat dry milk or BSA, the membranes were incubated overnight with selected primary antibodies: insulin receptor substrate 1 (IRS-1, 1:1000; Cell Signaling Technology, Danvers, MA, USA), phosphorylated insulin receptor substrate 1 (pIRS1 (Ser302), 1:1000; Cell Signaling), protein kinase B (Akt/PKB, 1:1000; Cell Signaling Technology), phosphorylated protein kinase B (pAkt/PKB (Ser473), 1:1000; Cell Signaling Technology), AS160 protein (AS160, 1:500; Cell Signaling Technology) phosphorylated AS160 protein (pAS160, 1:500; Cell Signaling Technology), glycogen synthase kinase 3 (GSK3, 1:500; Thermo Scientific, Rockford, IL, USA), phosphorylated glycogen synthase kinase 3 (pGSK3 (Ser9), 1:500; Thermo Scientific), glucose transporter 1 (GLUT1, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), glucose transporter 4 (GLUT4, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), pyruvate dehydrogenase (PDH, 1:5000; Abcam, Cambridge, UK), serine palmitoyltransferase, long chain base subunit 1 (SPTLC1, 1:500; Abcam), ceramide synthase 5 (LASS5, 1:500; Thermo Scientific), acid ceramidase (ASAH1, 1:500; Santa Cruz Biotechnology), sphingosine kinase 2 (SPHK2, 1:500; Sigma Aldrich, Saint Louis, MO, USA), cannabinoid receptor 1 (CB1, 1:500; Abcam), cannabinoid receptor 2 (CB2, 1:500; Abcam), transient receptor potential channel 1 (TRPV1, 1:500; Santa Cruz Biotechnology), and serotonin receptor (5-HT1A, 1:3000; Thermo Scientific). .. Next, nitrocellulose membranes were incubated with the corresponding secondary antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology).
Saline:Article Title: Chronic Cannabidiol Administration Attenuates Skeletal Muscle De Novo Ceramide Synthesis Pathway and Related Metabolic Effects in a Rat Model of High-Fat Diet-Induced Obesity
Article Snippet: .. After blocking in Tris-buffered saline with Tween-20 (TBST) with 5% non-fat dry milk or BSA, the membranes were incubated overnight with selected primary antibodies: insulin receptor substrate 1 (IRS-1, 1:1000; Cell Signaling Technology, Danvers, MA, USA), phosphorylated insulin receptor substrate 1 (pIRS1 (Ser302), 1:1000; Cell Signaling), protein kinase B (Akt/PKB, 1:1000; Cell Signaling Technology), phosphorylated protein kinase B (pAkt/PKB (Ser473), 1:1000; Cell Signaling Technology), AS160 protein (AS160, 1:500; Cell Signaling Technology) phosphorylated AS160 protein (pAS160, 1:500; Cell Signaling Technology), glycogen synthase kinase 3 (GSK3, 1:500; Thermo Scientific, Rockford, IL, USA), phosphorylated glycogen synthase kinase 3 (pGSK3 (Ser9), 1:500; Thermo Scientific), glucose transporter 1 (GLUT1, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), glucose transporter 4 (GLUT4, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), pyruvate dehydrogenase (PDH, 1:5000; Abcam, Cambridge, UK), serine palmitoyltransferase, long chain base subunit 1 (SPTLC1, 1:500; Abcam), ceramide synthase 5 (LASS5, 1:500; Thermo Scientific), acid ceramidase (ASAH1, 1:500; Santa Cruz Biotechnology), sphingosine kinase 2 (SPHK2, 1:500; Sigma Aldrich, Saint Louis, MO, USA), cannabinoid receptor 1 (CB1, 1:500; Abcam), cannabinoid receptor 2 (CB2, 1:500; Abcam), transient receptor potential channel 1 (TRPV1, 1:500; Santa Cruz Biotechnology), and serotonin receptor (5-HT1A, 1:3000; Thermo Scientific). .. Next, nitrocellulose membranes were incubated with the corresponding secondary antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology).
Incubation:Article Title: Chronic Cannabidiol Administration Attenuates Skeletal Muscle De Novo Ceramide Synthesis Pathway and Related Metabolic Effects in a Rat Model of High-Fat Diet-Induced Obesity
Article Snippet: .. After blocking in Tris-buffered saline with Tween-20 (TBST) with 5% non-fat dry milk or BSA, the membranes were incubated overnight with selected primary antibodies: insulin receptor substrate 1 (IRS-1, 1:1000; Cell Signaling Technology, Danvers, MA, USA), phosphorylated insulin receptor substrate 1 (pIRS1 (Ser302), 1:1000; Cell Signaling), protein kinase B (Akt/PKB, 1:1000; Cell Signaling Technology), phosphorylated protein kinase B (pAkt/PKB (Ser473), 1:1000; Cell Signaling Technology), AS160 protein (AS160, 1:500; Cell Signaling Technology) phosphorylated AS160 protein (pAS160, 1:500; Cell Signaling Technology), glycogen synthase kinase 3 (GSK3, 1:500; Thermo Scientific, Rockford, IL, USA), phosphorylated glycogen synthase kinase 3 (pGSK3 (Ser9), 1:500; Thermo Scientific), glucose transporter 1 (GLUT1, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), glucose transporter 4 (GLUT4, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), pyruvate dehydrogenase (PDH, 1:5000; Abcam, Cambridge, UK), serine palmitoyltransferase, long chain base subunit 1 (SPTLC1, 1:500; Abcam), ceramide synthase 5 (LASS5, 1:500; Thermo Scientific), acid ceramidase (ASAH1, 1:500; Santa Cruz Biotechnology), sphingosine kinase 2 (SPHK2, 1:500; Sigma Aldrich, Saint Louis, MO, USA), cannabinoid receptor 1 (CB1, 1:500; Abcam), cannabinoid receptor 2 (CB2, 1:500; Abcam), transient receptor potential channel 1 (TRPV1, 1:500; Santa Cruz Biotechnology), and serotonin receptor (5-HT1A, 1:3000; Thermo Scientific). .. Next, nitrocellulose membranes were incubated with the corresponding secondary antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology).
Article Title: Experimental Activation of Endocannabinoid System Reveals Antilipotoxic Effects on Cardiac Myocytes
Article Snippet: .. Next step involved overnight incubation of nitrocellulose membranes with selected primary antibodies, i.e., diacylglycerol acyltransferase 1 (DGAT1, 1:500, Novus Biologicals, USA), diacylglycerol acyltransferase 2 (DGAT2, 1:500, Santa Cruz Biotechnology, USA), adipose triglyceride lipase (ATGL, 1:1000, Santa Cruz Biotechnology, USA), glucose transporter 1 (GLUT1, 1:500, Santa Cruz Biotechnology, USA), glucose transporter 4 (GLUT4, 1:500, Santa Cruz Biotechnology, USA), insulin receptor substrate 1 (IRS-1, 1:1000; Cell Signaling Technology, UK), phosphorylated insulin receptor substrate 1 (pIRS1 (Ser302), 1:1000; Cell Signaling Technology, UK), protein kinase B (AKT/PKB, 1:1000; Cell Signaling Technology, UK), phosphorylated protein kinase B (pAKT/PKB (Ser473), 1:1000; Cell Signaling Technology, UK), glycogen synthase kinase 3 (GSK3, 1:500; Thermo Scientific, USA), phosphorylated glycogen synthase kinase 3 (pGSK3 (Ser9), 1:500, Thermo Scientific, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:500, Santa Cruz Biotechnology, USA), mitogen-activated protein kinase (MAPK; MAPK3/1, 1:1000; Cell Signaling Technology, UK), phosphorylated mitogen-activated protein kinase (pMAPK (Thr202/Tyr204); pMAPK3/1, 1:1000; Cell Signaling Technology, UK), citrate synthase (CS, 1:500, Santa Cruz Biotechnology, USA) and pyruvate dehydrogenase (PDH, 1:4000; Abcam, UK). ..
Article Title: Experimental Activation of Endocannabinoid System Reveals Antilipotoxic Effects on Cardiac Myocytes
Article Snippet: .. Next step involved overnight incubation of nitrocellulose membranes with selected primary antibodies, i.e., diacylglycerol acyltransferase 1 (DGAT1, 1:500, Novus Biologicals, USA), diacylglycerol acyltransferase 2 (DGAT2, 1:500, Santa Cruz Biotechnology, USA), adipose triglyceride lipase (ATGL, 1:1000, Santa Cruz Biotechnology, USA), glucose transporter 1 (GLUT1, 1:500, Santa Cruz Biotechnology, USA), glucose transporter 4 (GLUT4, 1:500, Santa Cruz Biotechnology, USA), insulin receptor substrate 1 (IRS-1, 1:1000; Cell Signaling Technology, UK), phosphorylated insulin receptor substrate 1 (pIRS1 (Ser302), 1:1000; Cell Signaling Technology, UK), protein kinase B (AKT/PKB, 1:1000; Cell Signaling Technology, UK), phosphorylated protein kinase B (pAKT/PKB (Ser473), 1:1000; Cell Signaling Technology, UK), glycogen synthase kinase 3 (GSK3, 1:500; Thermo Scientific, USA), phosphorylated glycogen synthase kinase 3 (pGSK3 (Ser9), 1:500, Thermo Scientific, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:500, Santa Cruz Biotechnology, USA), mitogen-activated protein kinase (MAPK; MAPK3/1, 1:1000; Cell Signaling Technology, UK), phosphorylated mitogen-activated protein kinase (pMAPK (Thr202/Tyr204); pMAPK3/1, 1:1000; Cell Signaling Technology, UK), citrate synthase (CS, 1:500, Santa Cruz Biotechnology, USA) and pyruvate dehydrogenase (PDH, 1:4000; Abcam, UK). ..
Phospho-proteomics:Article Title: Exercise prevents the adverse effects of maternal obesity on placental vascularization and fetal growth.
Article Snippet: Proteins were extracted from collected frozen placental tissues at −80°C using lysis buffer (100 mM Tris-HCL pH 6.8, 2.0% SDS, 20% glycerol, 0.02% Bromophenol Blue, 5% 2-mercaptoethanol, 100 mM NaF, and 1 mM Na 3 VO 4 ), and the protein concentration of lysates was determined by the Bradford Assay (Bio-Rad). .. The following antibodies were used for the detection of phosphorylation and total protein levels: phosphorylated insulin receptor substrate 1 [p-IRS-1 (Ser789); 2389S], insulin receptor substrate 1 (IRS-1; 3407S), p-Akt (Ser473; 9271), phosphorylated AMPKα [p-AMPKα (Thr172); 2535], AMPKα (2532), phosphorylated mammalian target of rapamycin [p-mTOR (Ser2448); 2971], p-4E-BP1 (Thr37/46; 9459), 4E-BP1 (9452), p-Erk1⁄2 (Thr202/Tyr204; 4370), Erk1⁄2 (4695), GLUT1 (12939), tumor necrosis factor α (TNF-α; 3707), interleukin-1β (IL-1β; 12242), and interleukin-6 (IL-6; 12153), all of which were purchased from Cell Signaling Technology (Danvers, MA, USA). .. Antibodies against Akt (sc-5298), mTOR (sc-517464), vascular endothelial growth factor (VEGF; sc-7269), endogenous ligand for the G-protein coupled APJ receptor (APLN; sc-293441), and hypoxia-inducible factor 1α (HIF1α; sc-13515) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). β-Actin and β-tubulin were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA, USA).
other:Article Title: SELENOM Knockout Induces Synaptic Deficits and Cognitive Dysfunction by Influencing Brain Glucose Metabolism.
Article Snippet: Antibodies Antibody name Cat Insulin receptor β Cell signaling #23413 Insulin receptor substrate-1 Cell signaling #3407 Phosphorylated insulin receptor substrate-1 (p-IRS-1) (Ser307) Cell signaling #2381 Anti-PI3K catalytic subunit gamma/PI3K-γ Abcam ab32089 Phosphorylated PI3K p85 (Tyr458)/p55 (Tyr199) Cell signaling #17366 Akt Cell signaling #9272 Phosphorylated Akt (Ser473) Cell signaling #4060 mTOR (7C10) Rabbit mAb Cell signaling #2983 Phosphorylated mTOR (Ser2448) Cell signaling #5536 p70 S6 kinase Cell signaling #34475 Phosphorylated p70 S6 kinase (Thr389) Cell signaling #97596 PSD95 Abcam ab238135 Synaptophysin Cell signaling #4329 SELENOM Sigma HPA019601 β-Actin Proteintech 66009-1-Ig GAPDH Proteintech 10494-1-AP α-Tubulin Proteintech 11224-1-AP HRP-linked anti-mouse IgG antibody Cell signaling #7076 HRP-linked anti-rabbit IgG antibody Cell signaling #7074
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