Phospho creb

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Phospho-CREB is a laboratory reagent used to detect and quantify the phosphorylated form of the cAMP Response Element Binding (CREB) protein. CREB is a transcription factor that plays a key role in cellular signaling pathways and gene expression regulation. The phosphorylation of CREB is a critical event in the activation of CREB-mediated transcription.

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3 protocols using phospho creb

Western Blot Analysis of Synaptic Proteins

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Total proteins were extracted using Buffer C lysis buffer. The protein concentrations were measured by bicinchoninic acid (BCA) assay. Forty micrograms of proteins were concentrated at 80 mV and separated at 100 mV using 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA) for 2 h at 300 mA following the electrophoresis. Membranes were blocked with 10% milk at room temperature for 1 h and incubated with the following primary antibodies: c-fos (sc-413, mouse monoclonal, Santa Cruz CA), BDNF (ab226843, rabbit polyclonal antibody; abcome echnology), CREB and phospho-CREB (sc-377154, sc-81486, mouse monoclonal, Santa Cruz, CA) at 4°C overnight. Membranes were washed 3 times (10 min × 3) with Tris-buffered saline-Tween (TBST) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Applygen, Beijing, China) for 1 h at room temperature followed by washing 3 times (10 min × 3) with Tris-buffered saline-Tween (TBST). Proteins were developed using an enhanced chemiluminescence (ECL) reagent kit (Applygen, Beijing, China) and radiographic films (Carestream, Xiamen, China). α-tubulin (ABT170, rabbit polyclonal, 1:10,000, Millipore, Temecula, USA) was used as the internal reference.

Li H., Wang T., Shi C., Yang Y., Li X., Wu Y, & Xu Z.Q. (2018). Inhibition of GALR1 in PFC Alleviates Depressive-Like Behaviors in Postpartum Depression Rat Model by Upregulating CREB-BNDF and 5-HT Levels. Frontiers in Psychiatry, 9, 588.

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Melanogenesis Regulation by Natural Compounds

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Sephadex LH-20 column of 25–10 μm was purchased from Pharmacia (Stockholm, Sweden), MCI-gel CHP 20P column of 75–150 μm was purchased from Mitsubishi (Tokyo, Japan), and YMC-gel ODS-A column of 230–70 and 500–400 mesh were purchased from YMC Co (PA, USA). Dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), L-DOPA, alpha-melanocyte stimulating hormone (α-MSH), and phosphate-buffered saline (PBS) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen–Gibco (Grand Island, NY, USA). Antibodies against MITF, tyrosinase, TRP-1, TRP-2, phospho-p38 MAPK, p38 MAPK, phospho-ERK, ERK, phospho-JNK, JNK, phospho-CREB, and CREB purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse and anti-goat IgG antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The cAMP assay kit was purchased from Cayman Chemical (Ann Arbor, MI). All other reagents were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA).

Jung H.G., Kim H.H., Paul S., Jang J.Y., Cho Y.H., Kim H.J., Yu J.M., Lee E.S., An B.J., Kang S.C, & Bang B.H. (2015). Quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside suppresses melanin synthesis by augmenting p38 MAPK and CREB signaling pathways and subsequent cAMP down-regulation in murine melanoma cells. Saudi Journal of Biological Sciences, 22(6), 706-713.

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Western Blot Analysis of Heart Proteins

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The heart tissue was homogenated in extraction buffer (20 mmol/L Tris HCl, 1 mmol/L Na 3 VO 4 , 5 mmol/L NaF). The heart protein was collected and subjected to 10% or 15% SDS-polyacrylamide gel electrophoresis then transferred to polyvinylidene difluoride membranes, which were blocked for 2 h with 5% non-fat milk in Tris-buffered saline (pH 7.4) containing 0.1% Triton X-100. The membranes were probed overnight at 4 C with the appropriate primary antibody as follows: total-p38, phospho-p38, and phospho-HSP27 (Cell Signaling Technology, Danvers, MA), total-p53, phospho-p53, total-CREB, phospho-CREB, Bax, Bcl2, Cytochrome c, caspase-3 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA). After washing and exposure to horseradish peroxidaseconjugated secondary antibody for 1 h at room temperature, antibody-antigen complexes were visualized by enhanced chemiluminescence assay. Bands corresponding to the protein of interest appeared as dark regions on the developed film. The film images of the Western blots were scanned and were analyzed using Image J (NIH image, National Institutes of Health, Bethesda, MD) analysis software (Tanno et al., 2003) . For quantitation of the proteins of interest, phosphorylated proteins were normalized to total protein expression.

Kumphune S., Surinkaew S., Chattipakorn S.C, & Chattipakorn N. (2015). Inhibition of p38 MAPK activation protects cardiac mitochondria from ischemia/reperfusion injury. Pharmaceutical biology, 53(12).

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