Anti total creb

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-total-CREB is a primary antibody that recognizes the CREB (cAMP response element-binding protein) protein, regardless of its phosphorylation state. CREB is a transcription factor that plays a crucial role in various cellular processes, including gene expression, cell growth, and survival. The Anti-total-CREB antibody can be used in applications such as Western blotting, immunohistochemistry, and immunocytochemistry to detect and quantify the CREB protein.

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6 protocols using anti total creb

Western Blot Analysis of Signaling Pathways

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After treatments, cells were washed with ice-cold PBS and lysed in Laemmli sample buffer. Whole-cell lysates were sonicated and heated to 95 °C for 5 min. Samples were resolved by SDS-PAGE and transferred onto 0.45 mm nitrocellulose membranes (Millipore) for immunoblotting. Membranes were blocked in TBS-Tween 20 (0.05%) containing 5% milk at room temperature for 1 h under shaking and probed overnight at 4 °C with the primary antibodies. The following antibodies were used: anti–phospho-ERK1/2 (E-4, sc-7383) from Santa Cruz Biotechnology; anti–total-ERK1/2, (9102, Cell Signaling), anti-phospho CREB (06-519, EMD Millipore), anti-total-CREB (9104, Cell Signaling), anti-phospho AKT (4058, Cell Signaling), anti-total-AKT (2920, Cell Signaling).
Signals were detected by HRP-conjugated secondary antibodies and enhanced chemiluminescence (SuperSignal West Dura, Pierce) using a GBOX Chemi XT4 (Syngene) or by IRDye700DX and IRDye800CW secondary antibodies (Rockland). Phosphorylation of MAPK and CREB was detected with the Odyssey Fc Imaging System (Li-Cor Biosystems). Phosphorylated proteins were relativized to its total protein level and results expressed as the percentage of maximum pERK1/2 after stimulation. Immunoreactive signals were analysed digitally using Fiji software.

Inda C., Bonfiglio J.J., dos Santos Claro P.A., Senin S.A., Armando N.G., Deussing J.M, & Silberstein S. (2017). cAMP-dependent cell differentiation triggered by activated CRHR1 in hippocampal neuronal cells. Scientific Reports, 7, 1944.

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Immunostaining of Stem Cell Markers

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OC explants or cells were fixed with 4% paraformaldehyde (Biosesang, Gyeonggi-do, Korea) for 15 min at room temperature and permeabilized using 0.2% Triton-X 100 in PBS (PBST). After blocking with 1% bovine serum albumin in 1× PBST, samples were incubated with primary antibodies at 4 °C for 24 h. The primary antibodies were anti-phospho-CREB (#9198, Cell Signaling), anti-total-CREB (#9197, Cell Signaling), anti-connexin 43 (#3512, Cell Signaling), and anti-SOX2 (1:500, Cell Signaling; #4900). Samples were washed thoroughly and incubated with secondary antibodies tagged with fluorescein isothiocyanate (FITC) or cyanine 3 (Cy3; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. F-actin was stained using phalloidin–Texas red (Invitrogen, Molecular Probes, Carlsbad, CA, USA), and nuclei were counterstained with 4′6,-diamidino-2-phenylindole (DAPI; Invitrogen). Coverslips were mounted onto slides with mounting medium (Vector Laboratories, Burlingame, CA, USA). The immunostained cells were observed using a Zeiss LSM 700 confocal microscope (Carl Zeiss Meditec, Jena, Germany).

Kim Y.J., Lee J.S., Kim H., Jang J.H, & Choung Y.H. (2021). Gap Junction-Mediated Intercellular Communication of cAMP Prevents CDDP-Induced Ototoxicity via cAMP/PKA/CREB Pathway. International Journal of Molecular Sciences, 22(12), 6327.

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Quantification of Insulin and cAMP Signaling

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Insulin was quantified in the media and lysate from cells cultured under the relevant conditions for 2 h (ELISA ALPCO, Salem, NH, cat. # 80-INSMSU-E01). Cyclic AMP was quantified by ELISA (Thermo Scientific, cat # EMSCAMPL) in lysates from MIN6 cells cultured for 20 h at low (3 mM) or high (25 mM) glucose, then exposed to high glucose ± exendin-4 (10 nM) for 10 min. Immunoblotting used the following antibodies: anti-phospho-CREB (Cell Signaling Technologies, cat # 4276, diluted 1:1000); anti-total CREB (Cell Signaling Technologies, cat # 4820, diluted 1:1000); anti-PKA-RIα (Cell Signaling Technologies, cat # 5675, diluted 1:1000); anti-PKA RIIα (BD Transduction Laboratories, cat # 612242, diluted 1:1000); anti-PKA-RIIβ (BD Transduction Laboratories, cat # 610625, diluted 1:1000); anti-PKA-Cα (BD Transduction Laboratories, cat # 610980, diluted 1:1000); anti-FLAG (Cell Signaling Technologies, cat # 8146, diluted 1:500); β-catenin (Cell Signaling Technologies, cat # 9562, diluted 1:1000); anti-tubulin (Cell Signaling Technologies, cat #5346, diluted 1:2000).

Rajan S., Dickson L.M., Mathew E., Orr C.M., Ellenbroek J.H., Philipson L.H, & Wicksteed B. (2015). Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A. Molecular Metabolism, 4(4), 265-276.

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Immunohistochemistry of Phosphorylated Proteins

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After deparaffinization and rehydration, antigen retrieval was performed using IHC-Tek ^™ Epitope Retrieval Solution (IHC WORLD, Ellicott City, MD, USA) and by boiling (95 °C) the slides in plastic Coplin jars for 20 min. After cooling at room temperature for 30 min, endogenous peroxidase was blocked in 3% H₂O₂ in methanol at room temperature for 10 min. The slides were incubated with 1% bovine serum albumin in PBST for 1 h. Anti-phospho-CREB (#9198, Cell Signaling), anti-total-CREB (#9197, Cell Signaling), anti-phospho-PKA (#9624, Cell Signaling), anti-Cx26 (51-2800, Zymed Laboratories), and anti-sodium potassium ATPase (ab7671, Abcam) antibodies were applied overnight at 4 °C. The slides were washed and incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature. The sections were washed three times with PBST buffer and reacted with 3,3-diaminobenzidine (DAB) using a DAB substrate kit (ab64238). Sections were counterstained with hematoxylin. Images were obtained by bright field microscopy (Olympus, Tokyo, Japan). Chromogen intensity was quantified as described [53 (link)]. The mean gray values in ROIs were averaged over five independent sections per group (n = 5) and presented as the relative chromogenic intensity compared with the controls.

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Western Blot Analysis of Signaling Proteins

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Protein samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), and blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBST) for 1 h. The membranes were incubated with primary antibodies at 4 °C overnight. After washing with PBST, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies (GenDepot, Katy, TX, USA) for 1 h at room temperature. Following washing of the membrane, the immunoblot bands were visualized using ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc, Grand Island, NY, USA). The following antibodies were used: anti-phospho-PKA substrate (#9624, Cell Signaling, Danvers, MA, USA), anti-cyclin D1 (#55506, Cell Signaling), anti-phospho-CREB (#9198, Cell Signaling), anti-total-CREB (#9197, Cell Signaling), anti-connexin 43 (#3512, Cell Signaling), anti-cleaved caspase-3 (#9661, Cell Signaling), and anti-cleaved PAPR (ab32064, Abcam, Cambridge, UK).

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Western Blot Analysis of Signaling Proteins

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After treatments, cells were washed with ice-cold PBS and lysed in Laemmli sample buffer. Whole-cell lysates were sonicated and heated to 95°C for 5 min. Samples were resolved by SDS-PAGE and transferred to 0.45-mm nitrocellulose membranes (EMD Millipore) for immunoblotting. Membranes were blocked in TBS–Tween 20 (0.05%) containing 5% milk at RT for 1 h under shaking and probed overnight at 4°C with the primary antibodies. The following antibodies were used: anti-sAC (R21; CEP Biotech), anti–c-Myc (9E10; sc-40) and anti–pERK1/2 (E-4; sc-7383) from Santa Cruz Biotechnology, Inc.; anti–total-ERK1/2 (9102; Cell Signaling Technology), anti-phospho CREB (06-519; EMD Millipore), and anti–total-CREB (9104; Cell Signaling Technology).
Chemiluminescent signals were detected by HRP-conjugated secondary antibodies and enhanced chemiluminescence (SuperSignal West Dura; Thermo Fisher Scientific) using a GBOX Chemi XT4 (Syngene). Phosphorylation of MAPK and CREB was detected with the Odyssey Fc Imaging System (LI-COR Biosciences), using anti-mouse-IRDye700DX and anti-rabbit-IRDye800CW secondary antibodies (Rockland). Phosphorylated proteins were relativized to the total protein level, and results were expressed as the percentage of maximum pERK1/2 after stimulation. Immunoreactive signals were analyzed digitally using ImageJ software (National Institutes of Health).

Inda C., dos Santos Claro P.A., Bonfiglio J.J., Senin S.A., Maccarrone G., Turck C.W, & Silberstein S. (2016). Different cAMP sources are critically involved in G protein–coupled receptor CRHR1 signaling. The Journal of Cell Biology, 214(2), 181-195.

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