Rabbit anti phospho creb

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-phospho-CREB is a primary antibody that specifically recognizes the phosphorylated form of the cyclic AMP response element-binding (CREB) protein. CREB is a transcription factor that plays a key role in regulating gene expression in response to various cellular signals.

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6 protocols using rabbit anti phospho creb

Immunostaining of Dorsal Root Ganglia

Cited 2 times

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DRGs were sectioned at a 20 µm thickness and processed for on-slide immunostaining. Primary antibodies used were mouse anti-CGRP (1:2000, Abcam), rabbit anti-phospho-CREB (1:1000, Cell Signaling), rabbit anti-phospho-Akt (1:500, Cell Signaling), and rabbit anti-PLC-γ (1:1500, Santa Cruz Biotechnology). The specificity of these antibodies had been carefully characterized in our previous studies.^{2 (link),13 (link)} The secondary antibodies used were Cy3- or Alexa 488-conjugated species-specific antibody. The final product of the slides was coverslipped with Citifluor (Citifluor Ltd., London) and viewed under a Zeiss fluorescent photomicroscope. Immunoreactive positive cells were counted in 6 to 10 sections randomly chosen from each ganglion and averaged as one sample. The area of section containing cells (excluding the area containing fibers) was selected using free-line tools integrated in the AxioVision measurement software (Carl Zeiss, Inc.) and was measured as mm². The number of positively stained cells was normalized against the measured area and expressed as number cells per mm². This method of quantification has been validated in our previous studies.^{13 (link)} To avoid double counting, we chose every third section for one specific antibody stained.

Hashmi F., Liu M., Shen S, & Qiao L.Y. (2016). Phospholipase C gamma mediates endogenous brain-derived neurotrophic factor-regulated calcitonin gene-related peptide expression in colitis-induced visceral pain. Molecular Pain, 12, 1744806916657088.

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Protein Expression Analysis in Adipocytes

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Cell were lysed in RIPA buffer supplemented with cOmplete® Protease Inhibitor Cocktail (Roche) and PhosSTOP® Phosphatase Inhibitor Cocktail (Roche). The lysates were centrifuged at 20,000g at 4 °C for 15 min, and the supernatants were incubated with DTT (100 mM) and NuPAGE® LDS Sample Buffer (Invitrogen) at 70 °C for 10 min. Proteins were separated on Novex® 4–12% Tris-Glycine Mini Gels (Invitrogen) and transferred to a PVDF membrane. The membrane was incubated with 5% skim milk at room temperature for 1 hr and subsequently with primary antibodies at 4 °C for overnight. For the detection of FABP7, the step of centrifuging cell lysates was omitted, and 5% BSA was used as a blocking solution. Primary antibodies were as follows and used at 1:1000 dilution unless otherwise stated: rabbit anti-FABP7 (#13347, Cell Signaling Technology), rabbit anti-UCP1 (U6382, Sigma Aldrich), rabbit anti-PGC-1α (1:200 v/v) (sc-13067, Santa Cruz Biotechnology), rabbit anti-PRDM16 (1:500 v/v) (ab106410, Abcam), rabbit anti-CREB (#9197, Cell Signaling Technology), and rabbit anti-phospho-CREB (#9198, Cell Signaling Technology). Appropriate secondary horseradish peroxidase-linked antibodies were used (Dako, UK). Immunoreactivity was detected with ECL Prime Western Blotting Detection Reagent (Amersham) and visualized using ImageQuant LAS 4000 mini (GE Healthcare).

Kawashima M., Bensaad K., Zois C.E., Barberis A., Bridges E., Wigfield S., Lagerholm C., Dmitriev R.I., Tokiwa M., Toi M., Papkovsky D.B., Buffa F.M, & Harris A.L. (2020). Disruption of hypoxia-inducible fatty acid binding protein 7 induces beige fat-like differentiation and thermogenesis in breast cancer cells. Cancer & Metabolism, 8, 13.

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Immunoblotting Analysis of Cell Signaling

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Immunoblotting analyses were performed using 25 μg total protein from the cell lysates. Protein concentrations was determined using the Bradford protein assay (Bio-Rad). Electrophoresis was performed using either an 8% or 12% SDS-PAGE gel and then subsequently transferred to PVDF membranes (Bio-Rad). The blots were and reacted with mouse anti-S100B (1:2000, BD Biosciences, Catalog No. 612376), rabbit anti-Phopho-STAT3 (1:1000, Cell signaling, Catalog No. 9145), rabbit anti-STAT3 (1:1000, Cell signaling, Catalog No. 12640), mouse anti-CREB (1:1000, Cell signaling, Catalog No. 9104), rabbit anti-Phospho-CREB (1:1000, Cell signaling, Catalog No. 9198), GAPDH (1:10,000, Cell signaling, Catalog No. 5174), β-actin (1:10,000, Sigma, Catalog No. A5541), rabbit anti-SP1 (1:1000, Cell signaling, Catalog No. 9389), rabbit anti-Phospho-MEK1/2 (1:1000, Cell signaling, Catalog No. 9121), mouse anti-vinculin (1:5000, Sigma, Catalog No. V9131); rabbit phospho-AKT (S473) (1:1000, Cell signaling Catalog No. 9271). Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher) was used at dilutions recommended by the manufacturer to detect proteins levels.

Alasady M.J., Terry A.R., Pierce A.D., Cavalier M.C., Blaha C.S., Adipietro K.A., Wilder P.T., Weber D.J, & Hay N. (2021). The calcium-binding protein S100B reduces IL6 production in malignant melanoma via inhibition of RSK cellular signaling. PLoS ONE, 16(8), e0256238.

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Comprehensive Antibody Characterization for Cellular Studies

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Our studies employed the following primary antibodies: Rabbit monoclonal anti-Engrailed (Monoclonal Antibody Facility, Institute of Neuroscience, University of Oregon), mouse polyclonal anti-Prox1 (AngioBio, 11-002P), mouse anti-FLAG M1 (prepared in-house, 1:5,000), rabbit anti-FLAG M2 (Sigma, F7425, 1:1,000), rabbit anti-GFP (which also detects YFP) (Thermo Fisher Scientific A11122, 1:5,000), mouse anti-PKA-C (BD Biosciences, 610980, 1:5,000), mouse anti-PKA-R (BD Biosciences, 610609, 1:500), mouse anti Arl13b (Antibodies, 75–287, 1:1,000), rabbit anti-RFP (Thermo Fisher Scientific, R10367, 1:1,000), rabbit anti-CREB (Cell Signaling Technology, 9197S, 1:1,000), and rabbit anti phospho-CREB (Cell Signaling Technology, 9198S, 1:1,000). For chemiluminescent western blots, HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Promega and used at 1:20,000. For infrared western blots, IR680- and IR800-conjguated secondary antibodies (LiCor) were used at 1:20,000. For immunofluorescence, AlexaFluor-conjugated secondary antibodies were obtained from Thermo Fisher Scientific and used at 1:1,000. M1 FLAG affinity resin and M1 FLAG-Alexa 647 conjugates were prepared in house.

Arveseth C.D., Happ J.T., Hedeen D.S., Zhu J.F., Capener J.L., Klatt Shaw D., Deshpande I., Liang J., Xu J., Stubben S.L., Nelson I.B., Walker M.F., Kawakami K., Inoue A., Krogan N.J., Grunwald D.J., Hüttenhain R., Manglik A, & Myers B.R. (2021). Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits. PLoS Biology, 19(4), e3001191.

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Quantitative Western Blot Analysis of Hippocampal Proteins

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We performed western blot experiments on hippocampal tissue lysates and protein levels were quantified after separation by acrylamide gel electrophoresis (gradient 12-7.5 %) and transfer to a nitrocellulose membrane, as previously described [19 (link), 72 (link)]. Membranes were incubated overnight at 4° C with the following primary antibodies: rabbit anti-phospho NMDA receptor 2A (1:250, Abcam), mouse anti- NMDA receptor 1 (1:2000, BD Bioscience), mouse anti- PSD95 (1:1000, Abcam), mouse anti-CREB (1:750, Cell Signaling) and rabbit anti-phospho CREB (1:1000, Cell Signaling), rabbit anti-CaMKII and rabbit anti-phospho CaMKII (1:1000, Cell Signaling), rabbit anti-TrkB (1:1000, Cell signaling), rabbit anti-BDNF (1:1000, Abcam) and mouse anti-alpha-tubulin (1:30000; Sigma-Aldrich, USA). Incubations with secondary antibodies were done for 2 hours at room temperature with anti-rabbit or anti-mouse immunoglobulin G (IgG) at 1:10000 dilutions (LI-COR Biosciences GmbH, Germany). Immunoreactivity was detected with LI-COR® Odyssey® system (LI-COR Biosciences, USA) by infrared fluorescence and quantified with ImageJ 1.48v software (NIH, MA, USA) by densitometry analysis of the immunoreactive bands.

Francesca E., Kristina J., María L.L., Sarah H., Jonas W., Angel C.M, & Maioli S. (2021). Long-term exposure to polypharmacy impairs cognitive functions in young adult female mice. Aging (Albany NY), 13(11), 14729-14744.

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Immunoblotting for Signaling Proteins

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The immunoblotting analyses were conducted as described previously [14] . The specimens were electrophoresed with 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes by electrophoresis. Following the blocking, the membranes were incubated at 4 °C overnight under gentle agitation with each primary antibody: goat anti-IL-1β (1:1000, R&D), mouse anti-Caspase-1 (1:1000; AdipoGen), goat anti-CatB (1:1000; Santa Cruz Biotechnology), mouse anti-IκBα (1:1000; Cell Signaling), rabbit anti-Synaptophysin (1:20000; Abcam), rabbit anti-Phospho-Akt (Thr308, 1:1000, Cell Signal), rabbit anti-Phospho-Akt (Ser473, 1:1000, Cell Signal), rabbit anti-total Akt (1:1000, Cell Signal), rabbit anti-Phospho-CREB (1:1000, Cell Signal), rabbit anti-total CREB (1:1000, Cell Signal). After washing, the membranes were incubated with HRP-labeled anti-goat (1:2000; GE Healthcare), anti-mouse (1:2000; R&D Systems), anti-rabbit (1:2000; GE Healthcare), and mouse anti-actin (1:5,000, Abcam) for 2 hours at room temperature. After that, the HRP-labeled protein bands were detected by an enhanced chemiluminescence detection system (ECL kit; GE Healthcare) with an image analyzer (LAS-1000; Fuji Photo Film).

Huang W., Zeng F., Gu Y., Jiang M., Zhang X., Yan X., Kadowaki T., Mizutani S., Kashiwazaki H., Ni J., & Wu Z. (2020). Leptomeningeal cells induce synaptic failure via Cathepsin B-mediated IL-1β production after Porphyromonas gingivalis infection.

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