Oleanolic acid (structure shown in Figure 1 ) was obtained from the Center for Drug Discovery at China Pharmaceutical University. Metformin (purity > 98%) was obtained from Sigma (St. Louis, MO, USA). The mouse insulin enzyme immunoassay ELISA kit was purchased from Invitrogen. Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) kits were purchased from Whitman Biotech (Nanjing, Ltd., China). Antibody (Ab) for G-6-Pase was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TORC2 was from Millipore (Billerica, MA, USA), and phospho-CREB and β-actin were from Abcam (Cambridge, MA, USA). The Abs against protein kinase B (PKB/Akt), phospho-Ser473-Akt (pAkt), PI3K, phospho-PI3K, AMPK, phosphor-AMPK, ACC, phosphor-ACC, PEPCK1, mTOR, and phospho-mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). The HRP-conjugated secondary Abs, affinity-purified mouse anti-rabbit IgG, and rabbit anti-mouse IgG were purchased from Sigma (St. Louis, MO, USA).
Phospho creb
Manufactured by Abcam
Sourced in United States, United Kingdom
Phospho-CREB is a lab equipment product that detects the phosphorylated form of the cAMP response element-binding (CREB) protein. CREB is a transcription factor that plays a key role in cellular processes such as memory formation, synaptic plasticity, and cell survival. The phosphorylation of CREB at specific serine residues is a crucial regulatory mechanism that modulates its activity and function.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (3 mentions)
Biotinylated secondary antibody, by Abcam (2 mentions)
Skim milk, by Merck Group (2 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (2 mentions)
Scopolamine hydrobromide, by Merck Group (2 mentions)
Avidin biotin peroxidase complex, by Abcam (2 mentions)
β actin, by Merck Group (2 mentions)
Phospho akt, by Abcam (2 mentions)
Hrp conjugated secondary ab, by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
Phosphor acc, by Cell Signaling Technology (1 mentions)
Rabbit anti mouse igg, by Merck Group (1 mentions)
Phosphor ampk, by Cell Signaling Technology (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Cresyl violet acetate, by Merck Group (1 mentions)
Copper 2 sulfate pentahydrate, by Merck Group (1 mentions)
Doublecortin dcx, by Abcam (1 mentions)
Bicinchoninic acid solution, by Merck Group (1 mentions)
7 protocols using phospho creb
1
Oleanolic Acid Modulates Glucose Metabolism
2
Neurobiological Assays in Cognitive Studies
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The following reagents were obtained from Sigma (St. Louis, MO, USA); acetic acid, bicinchoninic acid solution, bovine serum albumin, copper(II) sulfate pentahydrate, Cresyl violet acetate, 3,3′-diamino-benzidine (DAB), radioimmunoprecipitation assay (RIPA), scopolamine hydrobromide, skim milk, and 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate (THA). The CREB, phospho-CREB, NGF, BDNF, beta-actin, and secondary horseradish peroxidase (HRP)-conjugated antibodies used for western blotting were obtained from Abcam (Cambridge, MA) and Santa Cruz Biotechnology (Santa Cruz, CA); the doublecortin (DCX), biotinylated secondary antibodies, and avidin-biotin peroxidase complex used for immunohistochemical staining were obtained from Abcam (Cambridge, MA), Santa Cruz Biotechnology (Santa Cruz, CA) and Vector Laboratories (Burlingame, CA).
3
Polysaccharide Modulation of Microglia
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PSP contained 72.12% carbohydrate (Mw = 20.48 KDa) which was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Infrared spectroscopy showed that the PSP samples had typical polysaccharide structure characteristics.
The BV2 mouse microglial line was purchased from Wuhan Sunncell Bio-Technology Co., Ltd. (Wuhan, China); fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) from Gibco (Grand Island, NE, USA); lipopolysaccharides (LPS) from Sigma (St. Louis, MO, USA); enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor (TNF-α), interleukin (IL)-6, IL-1β, and IL-10 from Jianglai Biological Technology Co., Ltd. (Shanghai, China); and the Nitric Oxide (NO) assay kit, CCK-8 assay kit, and DCFH-DA Reactive Oxygen (ROS) Fluorescent Probe from Solarbio Science and Technology Co., Ltd. (Beijing, China). Antibodies targeting iNOS, BDNF, ERK, Phospho-ERK, CREB, Phospho-CREB, TrkB, and Phospho-TrkB from Abcam (Cambridge, UK); antibody targeting Arginase-1 from Cell Signaling Technology (Boston, MA, USA); antibodies targeting Iba-1, Notch1, and Hes1 from Santacruz Biotechnology (Santa Cruz, CA, USA); goat anti-rabbit AlexFluor 488® secondary antibodies from Servicebio Co., Ltd. (Shanghai, China); and CD16/CD32 monoclonal antibody PE and CD206 (MMR) monoclonal antibody PE from Ebioscience (San Diego, CA, USA).
The BV2 mouse microglial line was purchased from Wuhan Sunncell Bio-Technology Co., Ltd. (Wuhan, China); fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) from Gibco (Grand Island, NE, USA); lipopolysaccharides (LPS) from Sigma (St. Louis, MO, USA); enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor (TNF-α), interleukin (IL)-6, IL-1β, and IL-10 from Jianglai Biological Technology Co., Ltd. (Shanghai, China); and the Nitric Oxide (NO) assay kit, CCK-8 assay kit, and DCFH-DA Reactive Oxygen (ROS) Fluorescent Probe from Solarbio Science and Technology Co., Ltd. (Beijing, China). Antibodies targeting iNOS, BDNF, ERK, Phospho-ERK, CREB, Phospho-CREB, TrkB, and Phospho-TrkB from Abcam (Cambridge, UK); antibody targeting Arginase-1 from Cell Signaling Technology (Boston, MA, USA); antibodies targeting Iba-1, Notch1, and Hes1 from Santacruz Biotechnology (Santa Cruz, CA, USA); goat anti-rabbit AlexFluor 488® secondary antibodies from Servicebio Co., Ltd. (Shanghai, China); and CD16/CD32 monoclonal antibody PE and CD206 (MMR) monoclonal antibody PE from Ebioscience (San Diego, CA, USA).
4
Western Blot Analysis of Cell Signaling
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Cell lysates were harvested in radioimmune precipitation assay buffer containing 150 mM NaCl, 10 mM EDTA, 50 mM Tris‐HCl at pH 7.4, 1% NP‐40, 0.004% sodium azide, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Roche cOmplete™), which was supplemented with 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4. Whole cell lysate proteins were separated by SDS‐PAGE and electroblotted onto nitrocellulose membranes, which were incubated with several primary antibodies, including NFAT, phospho‐CREB (Abcam), phospho‐NF‐κB, NF‐κB, CREB, phospho‐Stat3, Stat3, phospho‐AKT, AKT, phospho‐p38, p38, phospho‐ERK, ERK (Cell Signaling), phospho‐JNK, JNK (Santa Cruz), and β‐actin (Sigma‐Aldrich). The immunocomplexes were then detected with horseradish peroxidase‐conjugated IgG (Jackson ImmunoResearch Laboratories), and the reaction was developed using an ECL detection kit (Amersham) in an ImageQuant LAS 4000 system (GE Healthcare Life Sciences).
5
Molecular Mechanisms of Memory Enhancement
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The reagents were obtained from Sigma (St. Louis, MO, USA); 3,3′-diamino-benzidine (DAB), reduced glutathione (GSH) radioimmunoprecipitation assay (RIPA), scopolamine hydrobromide, skim milk, 9-Amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate (tacrine), 1,1,3,3-tetraethoxypropane (TEP). The other reagents were obtained from the following vendors: CREB, phospho-CREB, BDNF, beta-actin, and secondary horseradish peroxidase (HRP)-conjugated antibodies for western blotting (Abcam, Cambridge, MA; and Santa Cruz Biotechnology; Santa Cruz, CA), doublecortin (DCX), 4-hydroxynonenal (4HNE), Ki67, biotinylated secondary antibodies, and avidin-biotin peroxidase complex (Abcam, Cambridge, MA; Santa Cruz Biotechnology, Santa Cruz, CA; and Vector, Burlingame, CA) for immunohistochemical staining, thiobarbituric acid (TBA; Lancaster Co., Lancashire, England), H2O2, (Junsei Chemical Co., Ltd., Tokyo, Japan).
6
Western Blot Analysis of Cerebellar Signaling Proteins
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The dissected cerebellum or cultured cerebellar slices were homogenized in ice-cold NP-40 lysis buffer (150 mM sodium chloride, 1.0% NP-40, 50 mM Tris pH 8.0) with freshly prepared protease and phosphatase inhibitors (Roche). Protein lysates with an equal amount were separated by SDS–PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membrane was blocked using 5% defatted milk powder and then incubated with primary antibodies (1:1000) against caspase-3 (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), phospho-ERK1/2 (Abcam), total-ERK1/2 (Cell Signaling Technology), phospho-p90RSK (Abcam), total-p90RSK (Abcam), phospho-Akt (Abcam), total-Akt (Cell Signaling Technology), phospho-cAMP response element binding (phospho-CREB; Abcam), or total-CREB (Abcam) overnight at 4°C, followed by incubation in HRP-conjugated anti-rabbit IgG antibody (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Signals were developed with Western Lightning plus-ECL Enhanced Chemiluminescence Substrate (PerkinElmer, Waltham, MA, USA) and the intensity was quantified by ImageJ 1.48 (National Institutes of Health, Bethesda, MD, USA). GAPDH served as the loading control.
7
Western Blot Analysis of Mitochondrial and Signaling Proteins
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Cell lysates were prepared in cell lysis buffer (Sigma) containing 50 mM sodium fluoride, 0.2 mM sodium orthovanadate, and 1% protease inhibitor. Total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk at room temperature for 1 h, the membranes were incubated with primary antibodies against UCP1 (Abcam, Cambridge, MA, USA), PGC-1α (Boster Bio, Pleasanton, CA), TFAM (Cell Signaling, Beverly, MA), NDUFB8 (Invitrogen, Carlsbad, CA, USA), SDHB (Invitrogen), UQCRC2 (Abcam), COXIV (Abcam), ATP5A (Abcam), p38 MAPK (Cell Signaling), phospho-p38 MAPK (Cell Signaling), CREB (Abcam), phospho-CREB (Abcam), AKT (Abcam), phospho-AKT (Abcam), phospho-AMPK (Cell Signaling), and β-actin (Sigma) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibodies at room temperature for 30 min. Membranes were developed with chemiluminescent HRP substrate (Advansta Inc, Menlo Park, CA, USA) using X-ray films.
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