P creb

Manufactured by Santa Cruz Biotechnology

Sourced in United States

P-CREB is a laboratory reagent that detects the phosphorylated form of the cAMP Response Element-Binding (CREB) protein. CREB is a transcription factor involved in various cellular processes. The phosphorylated form, P-CREB, is an indicator of CREB activation.

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Anti-P-CREB-Ser133 (2 citations) P-CREB (Ser133; (2 citations)

31 protocols using p creb

Immunocytochemistry of Metabolic Signaling

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Reagents and drugs: Antibodies used in this study were as follows: For immunocytochemistry first antibodies: goat GHSR1a, mouse H1R, rabbit NPY (all from Santa Cruz Biotechnologies; dilution factor 1:250), Mouse GAD-65 (Chemicon 1:250); Secondary antibodies: Donkey antigoat-Alexa Fluor 488, Donkey anti-rabbit-Alexa Fluor 488, Donkey-anti-mouse-Alexa Fluor 555, Donkey-anti-goat-Alexa Fluor 647, Donkey-anti-rabbit-Alexa Fluor 568 all from pCREB (Santa Cruz Biotechnologies; dilution factor 1:250 IF, 1:1000 WB), pAMPK, AMPK, FOXO1 (Cell Signaling Technology 1:1000), UCP2 (Santa Cruz Biotechnologies; dilution factor 1:1000 ).
Secondary antibodies for WB were anti-rabbit (for AMPK, pAMPK, FOXO1 and pCREB) or anti-goat (for UCP-2) IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnologies, USA; dilution factor 1:5000). 2-(3-trifluoromethylphenyl) histamine (FMPH), Pyrilamine (PYR) and Olanzapine were obtained from Sigma-Aldrich (Sigma, NSW, Australia).

Chen X., Yu Y., Zheng P., Jin T., He M., Zheng M., Song X., Jones A, & Huang X.F. (2020). Olanzapine increases AMPK-NPY orexigenic signaling by disrupting H1R-GHSR1a interaction in the hypothalamic neurons of mice. Psychoneuroendocrinology, 114.

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Berberine Hydrochloride Signaling Pathway

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Berberine hydrochloride (purity ≥98%) was purchased from Dalian Meilun Biotechnology Co., Ltd. and dissolved in double‐distilled water. Quinolinic acid was purchased from Sigma‐Aldrich. Go6983 (a pan‐PKC inhibitor) was purchased from Selleck Chemicals. The rabbit polyclonal antibodies against GSK3β, p‐GSK3β(S9) and p‐PKCζ(T410) were purchased from Bioworld. The rabbit polyclonal antibodies against PKCζ were purchased from Proteintech. The rabbit polyclonal antibodies against ERK1/2, p‐ERK1/2, CREB, p‐CREB and BDNF, along with the mouse polyclonal antibodies against β‐actin and secondary antibodies, were purchased from Santa Cruz.

Liu P., Li Y., Qi X., Xu J., Liu D., Ji X., Chi T., Liu H, & Zou L. (2019). Protein kinase C is involved in the neuroprotective effect of berberine against intrastriatal injection of quinolinic acid‐induced biochemical alteration in mice. Journal of Cellular and Molecular Medicine, 23(9), 6343-6354.

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Immunohistochemical Analysis of Trigeminal Ganglion

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After undergoing behavioral testing, 5–8 rats per group were selected by randomization and assigned to subsequent IHC detection. Rats were anaesthetized with ketamine and xylazine (i.p.), and then the whole brain was removed and fixed in 5% paraformaldehyde for 3 h. The transverse sections of trigeminal ganglion samples were incubated in 0.3% Triton X-100 and 2% normal goat serum for 1 h, followed by incubation with antibodies against BDNF, TrkB, p-CREB, and p-Erk (Santa Cruz Biotechnology). Following incubation, sections were thoroughly washed with PBS and incubated with biotinylated secondary antibody and avidin-biotin complex (Vectastain – ABC kit PK-6100 Elite, 1 h) for color detection.

Guo J.Q., Deng H.H., Bo X, & Yang X.S. (2016). Involvement of BDNF/TrkB and ERK/CREB axes in nitroglycerin-induced rat migraine and effects of estrogen on these signals in the migraine. Biology Open, 6(1), 8-16.

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Protein Isolation and Western Blot Analysis

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The proteins were isolated from the cells using a radioimmunoprecipitation assay (RIPA) buffer along with a protease inhibitor cocktail, 2 mM phenylmethylsulphonyl fluoride (PMSF), and 1 mM sodium orthovanadate. The cell lysates were centrifuged at 16,000× g for 20 min at 4 °C and the protein was quantified. Equal amounts of proteins were resolved by SDS-PAGE using 6–15% resolving gels and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% normal horse serum (NHS) in TBS-T and then incubated overnight at 4 °C with primary antibodies specific for NR2B (1:200; Santa Cruz Biotechnology), p-CREB (1:500; Santa Cruz Biotechnology), CREB (1:500; Santa Cruz Biotechnology), cleaved caspase-3 (1:150; Merck Millipore, CA, USA), p53 (1:500; Santa Cruz Biotechnology), and GAPDH (1:1000; Santa Cruz Biotechnology) as well as horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat (Santa Cruz Biotechnology). The protein bands were visualized by enhanced chemiluminescence detection (GE Healthcare, Buckinghamshire, UK) and quantified using ImageJ software 1.53t, Bethesda, MD, USA). All the bands were normalized with GAPDH and p-CREB was normalized with CREB.

Song A., Cho G.W., Moon C., Park I, & Jang C.H. (2022). Protective Effect of Resveratrol in an Experimental Model of Salicylate-Induced Tinnitus. International Journal of Molecular Sciences, 23(22), 14183.

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Antioxidant and Neuroprotective Assays

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Organic solvents were purchased from J.T. Baker (Ulsan, Korea). Dulbecco’s modified Eagle’s medium (DMEM), DMEM with F-12 Ham 1:1 mixture (DMEM/F-12), fetal bovine serum (FBS), and horse serum were procured from Hyclone (South Logan, UT, USA). Hydrogen peroxide (H₂O₂), ascorbic acid, dimethyl sulfoxide (DMSO), dichlorofluorescein acetate (DCF-DA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were purchased from Sigma-Aldrich (St. Luis, MO, USA). RNA-Bee reagent and GoTaq^®® Green Master Mix were purchased from AMS Bio (Abingdon, UK) and Promega (Madison, WI, USA), respectively. All the primary antibodies that recognized glutathione S-transferase (GST) m5, glutathione peroxidase (GPX) 4, peroxiredoxin (PRX) 3, phosphorylated cAMP response element-binding protein (P-CREB), nectin-2 and β-actin were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Kopalli S.R., Yoo S.K., Kim B., Kim S.K, & Koppula S. (2022). Apigenin Isolated from Carduus crispus Protects against H2O2-Induced Oxidative Damage and Spermatogenic Expression Changes in GC-2spd Sperm Cells. Molecules, 27(6), 1777.

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Curcumin-mediated NF-κB Pathway Regulation

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Curcumin and propidium iodide (PI) were purchased from Sigma-Aldrich (Germany). All antibodies (PARP, lamin B, caspase-3, pCREB, catalytic PKA subunits, Ras GAP, pRaf, pErk, NF-κB subunits p50 and p65, RelB, IκBα, actin, and proliferating cell nuclear antigen (PCNA) were obtained from Santa Cruz Biotechnology (USA) and Cell Signaling (USA). Electrophoresis reagents and Bio-Rad protein assay kit were purchased from Bio-Rad Laboratories (USA). The 22-mer double-stranded NF-κB oligonucleotides were obtained from Promega (USA). Dichlorofluorescein diacetate (DCFHDA) was obtained from Molecular Probes (USA). PGE₂, hydroxy PGE₂, and butaprost were purchased from Cayman Chemicals (USA). H89 and PD98059 were obtained from Sigma Aldrich and Cell Signaling. Nitrocellulose membrane and X-ray reagents were purchased from (Amersham Pharmacia Biotech, UK). These chemicals were used according to the manufacturer’s instructions.

Shehzad A., Islam S.U., Lee J, & Lee Y.S. (2014). Prostaglandin E2 Reverses Curcumin-Induced Inhibition of Survival Signal Pathways in Human Colorectal Carcinoma (HCT-15) Cell Lines. Molecules and Cells, 37(12), 899-906.

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Quantitative Analysis of Hippocampal Proteins

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After anesthesia with Zoletil, brains (n=6 per group) were quickly removed from the skull and both hippocampi were dissected and frozen. Tissue lysis for protein extraction was performed as previously described [16 (link)]. Protein concentrations were quantified with the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA) and samples were stored at −80℃. Hippocampal lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrophoretic transfer onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Proteins were immunoblotted with antibodies to glial fibrillary acidic protein (GFAP, 1:3000; Sigma-Aldrich, St. Louis, MO, USA), pCREB (1:1000; Santa Cruz Biotech, Santa Cruz, CA, USA), CREB (1:1000; Santa Cruz Biotech), LCN2 (1:1000; R&D Systems, Minneapolis, MN, USA), COX-2 (1:1000; Santa Cruz Biotech), and transforming growth factor-β1 (TGF-β1, 1:1000; Santa Cruz Biotech). To determine relative protein amounts, β-actin (1:30000; Sigma) was used as an internal control. Specific proteins were visualized using an ECL substrate (Pierce, Rockford, IL, USA). The Multi Gauge v 3.0 image analysis program (Fujifilm, Tokyo, Japan) was used to measure band intensity by densitometry.

Park Y.S., Jin Z., Jeong E.A., Yi C.O., Lee J.Y., Park I.S, & Roh G.S. (2017). Cilostazol attenuates kainic acid-induced hippocampal cell death. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 22(1), 63-70.

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Immunohistochemical Staining of Brain Tissue

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Frozen sections (30-μm thickness) were incubated with a blocking buffer (1× PBS/5% normal goat serum/0.3% Triton X-100) for 1 h. Sections were incubated with primary antibodies overnight in an antibody dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) at 4 °C. The antibodies were as follows: BrdU (Cat. OBT0030G, AbD Serotec, Oxford, UK), NeuN (Cat. MAB377; ABN78, Millipore Corporation, Billerica, MA, USA), Syn1 (Cat. ab64581, Abcam, Cambridge, UK), TH (Cat. AB152, Millipore Corporation), and pCREB (Cat. sc-7978-R, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were incubated with a fluorescent secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA) for 2 h and 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen Corporation, Carlsbad, CA, USA) for 30 min in the dark, respectively. After slide mounting with a mounting medium (Vector Laboratories, Inc.), images were captured using a fluorescence microscope (Carl Zeiss, Inc.).

Kim Y.R., Kim H.N., Hong K.W., Shin H.K, & Choi B.T. (2017). Antidepressant Effects of Aripiprazole Augmentation for Cilostazol-Treated Mice Exposed to Chronic Mild Stress after Ischemic Stroke. International Journal of Molecular Sciences, 18(2), 355.

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Western Blot Analysis of Signaling Proteins

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Cells were lysed in 0.5% Nonidet P-40 lysis buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Following electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane, Immobilon-P (Millipore Co., Milford, MA, USA). Membranes were blocked with Tris-buffered saline-Blotto/Blotto B (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour and subsequently incubated overnight with antibodies directed against α-CaMKII, p-α-CaMKII, p-CREB, CREB, p-c-Jun, c- Fos, Lamin B1, HIF-1α, VEGFR-1, VEGFR-2 or β-actin (Santa Cruz Biotechnology and Cell Signaling Technology, Beverly, MA, USA). Signals were detected using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (ECL; Amersham Biosciences, Pittsburgh, PA, USA) [14 (link)]. Band density was measured using ImageJ software and normalized to β-actin.

Daft P.G., Yang Y., Napierala D, & Zayzafoon M. (2015). The Growth and Aggressive Behavior of Human Osteosarcoma Is Regulated by a CaMKII-Controlled Autocrine VEGF Signaling Mechanism. PLoS ONE, 10(4), e0121568.

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Western Blot Analysis of CREB and P-CREB

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Whole cell extracts (30 μg) were separated on a 13% Sodyum-Dodecyl-Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE) according to [44 (link)], and transferred onto a nitrocellulose membrane. Western blot analysis was performed by using specified primary antibodies against cyclic AMP-responsive element binding protein (CREB) and phosphorylated-CREB (P-CREB) (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), according to the manufacturer’s suggested concentrations. Protein loading was assessed by reprobing the blots with GAPDH (1:3000; Santa Cruz Biotechnology). After incubation with the corresponding horseradish peroxidase-conjugated secondary antibody (1:3000; Cell Signaling Technology, Danvers, MA, USA) signals were developed using an enhanced chemiluminescence kit (ClarityTM Western ECL Substrate, Bio-Rad), acquired by ChemiDoc Imaging System XRS (BioRad), and analyzed for densitometry with the Image J Lab software 1.8.0_112 (https://imagej.nih.gov/ij/index.html).

Tanzarella P., Ferretta A., Barile S.N., Ancona M., De Rasmo D., Signorile A., Papa S., Capitanio N., Pacelli C, & Cocco T. (2019). Increased Levels of cAMP by the Calcium-Dependent Activation of Soluble Adenylyl Cyclase in Parkin-Mutant Fibroblasts. Cells, 8(3), 250.

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