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Total creb

Manufactured by Cell Signaling Technology
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Total CREB is a lab equipment product that measures the total amount of the CREB (cAMP response element-binding) protein in a sample. CREB is a transcription factor that plays a crucial role in various cellular processes, including gene expression, neuronal function, and cellular metabolism. The Total CREB product provides a quantitative assessment of the total CREB levels in a given sample, without making any interpretations or claims about its intended use.

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Lab products found in correlation

Phospho creb, by Cell Signaling Technology (6 mentions) P creb, by Cell Signaling Technology (5 mentions) Total erk1 2, by Cell Signaling Technology (4 mentions) Total erk, by Cell Signaling Technology (3 mentions) Total akt, by Cell Signaling Technology (3 mentions) Total p38, by Cell Signaling Technology (3 mentions) Triton x 100, by Merck Group (2 mentions) Anti β actin, by Merck Group (2 mentions) Total gsk3β, by Cell Signaling Technology (2 mentions) Phosphor creb, by Cell Signaling Technology (2 mentions) Phospho erk, by Cell Signaling Technology (2 mentions) Phospho creb ser133, by Cell Signaling Technology (2 mentions) Sucrose, by Thermo Fisher Scientific (1 mentions) Mgcl2, by Thermo Fisher Scientific (1 mentions) Sytox green nucleic acid stain, by Thermo Fisher Scientific (1 mentions) Sodium azide, by Merck Group (1 mentions) Hepes buffer, by Corning (1 mentions) 12 o tetradecanoylphorbol 13 acetate, by Merck Group (1 mentions) Anti rabbit igg pi 1000, by Vector Laboratories (1 mentions) Mushroom tyrosinase, by Merck Group (1 mentions)

Close spelling variants of this product

Anti-total-CREB (6 citations) Rabbit anti-total CREB (2 citations) Total-CREB (#9197) (2 citations)

24 protocols using total creb

1

Isolation and Analysis of Transcription Factors in CD4+ T Cells

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CD4+ T cells were isolated from B6 or SLE123 spleens via negative MACS. These cells were then rested in DMEM with 5% FBS, Pen-Strep, and 2-ME for 1 hour. They were then stimulated as described. These cells were then resuspended in a buffer containing 320 mM sucrose (Thermo Fisher Scientific), 10 mM HEPES buffer (Corning), 8 mM MgCl2 (Invitrogen), Roche EDTA-free Complete Protease Inhibitor, and 0.1% Triton X-100 (MilliporeSigma) for 15 minutes on ice. The cells were then fixed for 30 minutes on ice with 4% PFA (Pierce) added to the previously described buffer. Cells were then washed and permeabilized with 0.3% Triton X-100 on ice for 15 minutes. Isolated nuclei were then washed with FACS buffer containing 3% FBS and sodium azide (MilliporeSigma). They were then stained with SyTOX Green Nucleic Acid Stain (Thermo Fisher Scientific), and total CREB (Cell Signaling Technology, 48H2), total ATF1 (MilliporeSigma, SAB4501950), or total CRTC2 (MilliporeSigma, 5B10). Individual samples were acquired on a BD LSR Fortessa Flow Cytometer and analyzed with FlowJo software (TreeStar).
Wilson C.S., Stocks B.T., Hoopes E.M., Rhoads J.P., McNew K.L., Major A.S, & Moore D.J. (2021). Metabolic preconditioning in CD4+ T cells restores inducible immune tolerance in lupus-prone mice. JCI Insight, 6(19), e143245.
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2

Fucoidan and L-DOPA Melanogenesis Regulation

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Fucoidan (from Fucus vesiculosus), 3,4-dihydroxyl-L-phenylalanine (L-DOPA), cholera toxin (CT), 12-O-tetradecanoylphorbol-13-acetate (TPA), and mushroom tyrosinase were from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies specific for phospho-ERK1/2 (Thr202/Tyr204, #9101S), total (phosphorylated and non-phosphorylated) ERK1/2 (#9102), phospho-specific Akt (Ser473, #9271), total Akt (#9916), phospho-CREB (ser133, #9198), and total-CREB (#9197) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for tyrosinase (C-19) and actin (I-19) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Microphthalmia Ab-1 (C5, MS-771-P0) was from NeoMarkers (Fremont, CA, USA). PD98059 was from Cell Signaling Technology. Secondary antibodies specific for anti-goat IgG (PI-9500), anti-mouse IgG (PI-2000), and anti-rabbit IgG (PI-1000) were purchased from Vector Laboratories (Burlingame, CA, USA).
Song Y.S., Balcos M.C., Yun H.Y., Baek K.J., Kwon N.S., Kim M.K, & Kim D.S. (2014). ERK Activation by Fucoidan Leads to Inhibition of Melanogenesis in Mel-Ab Cells. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 19(1), 29-34.
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3

Signaling Pathway Analysis Protocol

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Western blot, cell fractionation and Co-IP analysis were carried out as described in our previous publications.23 (link),26 (link),29 (link) The following primary antibodies were used: anti-ITGA9, anti-CD31 (Abcam, Cambridge, MA); anti-β-catenin, anti-non-phospho-β-catenin, phospho-β-catenin (Ser33/37/Thr41, Thr41/Ser45, Ser675), phospho-GSK3α (Ser21), phospho-GSK3β (Ser9), total GSK3α, total GSK3β, phosphor-CREB (Ser133), total CREB, integrin-linked kinase (ILK), PKA-Cα, and PARP-1 (Cell Signaling Technology, Beverly, MA); anti-PKA RIIα regulatory unit (Santa Cruz Biotechnology, Dallas, TX); anti-β-actin (Sigma, St. Louis, MO).
Wang Z., Li Y., Xiao Y., Lin H.P., Yang P., Humphries B., Gao T, & Yang C. (2019). Integrin α9 depletion promotes β-catenin degradation to suppress triple negative breast cancer tumor growth and metastasis. International journal of cancer, 145(10), 2767-2780.
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4

Melanocyte Protein Expression Analysis

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In this experiment, the following antibodies were used: tyrosinase, Tyrp1, DCT, MITF, SOX10, Pmel, Ki67, and PCNA were obtained from Abcam (Cambridge, UK). Total ERK, phospho-ERK, total CREB, and pCREB were purchased from Cell Signaling Technology (Danvers, MA, USA). Another antibody for tyrosinase (Santa Cruz, Dallas, TX, USA) was also used to confirm bands. Antibody for MLANA was obtained from Cell Marque (Rocklin, CA, USA). α-tubulin (Gentex, Holland, MI, USA) and HSP90 (Santa Cruz) were used as internal loading controls. Secondary antibodies used for western blotting were as follows: goat anti-rabbit IgG- horseradish peroxidase (HRP) (1:5,000), goat anti-mouse IgG-HRP (1:5,000), and mouse anti-goat IgG-HRP (1:5,000).
Yoo H., Lee H.R., Kim K.H., Kim M.A., Bang S., Kang Y.H., Kim W.H., Song Y, & Chang S.E. (2021). CRTC3, a sensor and key regulator for melanogenesis, as a tunable therapeutic target for pigmentary disorders. Theranostics, 11(20), 9918-9936.
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5

Immunolabeling of Neural Progenitor and Granule Cells

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CGNPs and NPCs were fixed with 4% formaldehyde (Sigma), permeabilized with 0.1% Triton X-PBS, and blocked with 5% normal goat serum (Thermo Fisher). Primary antibodies were GFAP (1:400) (Dako) and Nestin (1:1000) (BD Biosciences) for NPCs, and phospho-CREB (1:1000) (Cell Signaling), NeuN (1:1000), Doublecortin (1:500) (Abcam) for CGNPs. Secondary antibodies were Alexa Fluor 488 or Alexa Fluor 568 (1:500) (Invitrogen). Cells were counterstained with DAPI and imaged on a Leica inverted fluorescence microscope.
Whole brains from P7 and P30 wild-type C57/BL6 mice were fixed with 4% formaldehyde. Brains were cryoprotected with a sucrose gradient (10%, 20% and 30% sucrose in PBS) and sagitally embedded in Tissue-Tek OCT (Sakura). 6–10 μm cryosections were generated on a Leica cryostat. Antigen retrieval was performed using Citrate buffer (100 mM, pH 6.0). Immunolabelings were performed as for NPCs and CGNPs. Primary antibodies were phospho-CREB (1:1000), total CREB (1:1000) (Cell Signaling), BMP6 (1:1000) and BMP12 (1:1000) (Abcam). Slides were mounted using Vectashield (Vector Laboratories), imaged on a Leica TCS SP8 confocal microscope and analysed using Fiji22 (link).
Armandari I., Zomerman W.W., Plasschaert S.L., Smit M.J., Martini T.E., de Camargo Magalhães E.S., Hogeling S.M., Rozema-Huizinga G.C., Lourens H.J., Meeuwsen-de Boer T.G., Scherpen F.J., de Bont E.S, & Bruggeman S.W. (2021). CREB signaling activity correlates with differentiation and survival in medulloblastoma. Scientific Reports, 11, 16077.
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6

Effect of Melanogenesis Modulators on Skin Pigmentation

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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco BRL (Carlsbad, CA, USA). OEA, kojic acid, MK886, L-DOPA, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), α-melanocyte-stimulating hormone (α-MSH), PD98059 and PTU were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Antibodies against MC1R (1:1000, Cell Signaling Technology, Boston, USA), MITF (1:1000, Cell Signaling Technology), TRP-1 (1:1000, Cell Signaling Technology), Tyrosinase (1:1000, Cell Signaling Technology), PPARα (1:1000, Abcam, Cambridge, UK), total-ERK (1:1000, Cell Signaling Technology), phospho-ERK (Thr202/Thr204) (1:1000, Cell Signaling Technology), total-Akt (1:1000, Cell Signaling Technology), phospho-Akt (Ser473) (1:1000, Cell Signaling Technology), total-CREB (1:1000, Cell Signaling Technology), phospho-CREB (Ser133) (1:1000, Cell Signaling Technology), total-p38 (1:1000, Cell Signaling Technology), phospho-p38 (1:1000, Cell Signaling Technology) and mouse monoclonal anti-β-actin (1:10000, Sigma) were obtained from the indicated manufacturers. OEA, kojic acid, MK886, PD98059 and PTU were dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration was less than 0.05%. L-DOPA was dissolved in phosphate buffered saline (PBS).
Zhou J., Ren T., Li Y., Cheng A., Xie W., Xu L., Peng L., Lin J., Lian L., Diao Y., Jin X, & Yang L. (2017). Oleoylethanolamide inhibits α-melanocyte stimulating hormone-stimulated melanogenesis via ERK, Akt and CREB signaling pathways in B16 melanoma cells. Oncotarget, 8(34), 56868-56879.
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7

Protein Extraction from Liver Samples

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Livers were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer [50 mM tris (pH 8), 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, and 1% NP-40] containing proteases and phosphatases inhibitors (1× cOmplete EDTA free cocktail tablet, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, and 100 μM C3H7Na2O6P) (Sigma-Aldrich, St. Louis, MO). Samples were nutated for 20 min at 4°C and sonicated briefly. Cell lines and PH were washed with 1× phosphate-buffered saline, harvested in RIPA lysis buffer, rocked, and briefly sonicated. Samples were centrifugated at top speed for 10 min at 4°C and the supernatant was collected. Protein concentrations were measured using Bradford reagent and a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Indianapolis, IN). Ten to fifty micrograms of lysate was resolved on SDS–polyacrylamide gel electrophoresis gels. Antibodies used for Western blots were REV-ERBα, phospho-CREB, total CREB (Cell Signaling Technology, Danvers, MA), BMAL1, TATA box–binding protein (Abcam, Cambridge, UK), CRY1 (Bethyl Laboratories, Montgomery, TX), and α-TUBULIN (Sigma-Aldrich, St. Louis, MO).
Verlande A., Chun S.K., Goodson M.O., Fortin B.M., Bae H., Jang C, & Masri S. (2021). Glucagon regulates the stability of REV-ERBα to modulate hepatic glucose production in a model of lung cancer–associated cachexia. Science Advances, 7(26), eabf3885.
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8

Quantifying mRNA and Protein Expression

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Total RNA from cultured cells or tissues was purified using TRIzol (Invitrogen) for cDNA synthesis (ABI High Capacity Reverse Transcription Kit). Relative mRNA expression was quantified by qPCR using SYBR Green dye (ABI) and specific primers (see Table S1 for primer sequences). For Western Blotting, whole cell lysates were prepared with RIPA buffer, separated by SDS-PAGE and transferred to ImmobilonP membranes (Millipore). The following antibodies were used: anti-CLK2 (NeoBioLab), anti-UCP1 (Abcam), pCREB (Cell Signaling), total CREB (Cell Signaling), anti-Tubulin (Milipore), anti-Lamin (Abcam), anti-Actin (Cell Signaling), anti-Porin (Abcam) anti-pAKT S473 (Cell Signaling), and anti-Pan AKT (Cell Signaling).
Hatting M., Rines A.K., Luo C., Tabata M., Sharabi K., Hall J.A., Verdeguer F., Trautwein C, & Puigserver P. (2017). Adipose tissue CLK2 promotes energy expenditure during high fat diet intermittent fasting. Cell metabolism, 25(2), 428-437.
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9

Flow Cytometry Antibodies for Immune Profiling

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Flow cytometry antibodies to CD19 (clone eBio1D3), IL-10 (clone JEs5-16E3), TNFα (clone MP6-XT22), IgM (clone 11/41), and IgD (clone 11-26c.2a) were from eBiosciences; F4/80 (clone BM8), CD1d (clone 1B1), and CD5 (clone 53-7.3) were from BioLegend; IL-6 (clone MP5-20F3) was from BD Biosciences; anti-rabbit IgG Fab2–PE conjugate was from Cell Signaling Technology; and rat anti-mouse CD16/CD32 Fc blockTM (clone 2.4G2) was from BD PharmingenTM. Western blotting antibodies to total MSK1 and total RSK2 were made in-house. Phospho-ERK1/2 (Thr202/Tyr204) (catalog no. 9101), total ERK1/2 (catalog no. 9102), phospho-p38 (Thr180/Tyr182) (catalog no. 4511), total p38 (catalog no. 9212), phospho-CREB (Ser133)/phospho-ATF1 (Ser63) (catalog no. 9198), total CREB (catalog no. 9197), phospho-MSK1 (Thr581) (catalog no. 9595), phospho-GSK3α/β (S21/S9) (catalog no. 9331), phospho-RSK (Thr359/Ser363 numbered for human RSK1) (catalog no. 9344), and total GAPDH (catalog no. 2118) were from Cell Signaling Technology.
Sutavani R.V., Phair I.R., Barker R., McFarlane A., Shpiro N., Lang S., Woodland A, & Arthur J.S. (2017). Differential control of Toll-like receptor 4–induced interleukin-10 induction in macrophages and B cells reveals a role for p90 ribosomal S6 kinases. The Journal of Biological Chemistry, 293(7), 2302-2317.
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10

Immunocytochemical Profiling of Glial Cells

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Immunocytochemistry was performed as described earlier (20 (link)). Briefly, 8-well chamber slides containing BV-2 microglial cells, mouse primary microglia or astrocytes cultured to 70–80% confluence were fixed with chilled methanol (Fisher Scientific, Waltham, MA) overnight, followed by two brief rinses with filtered PBS. Samples were blocked with 2% BSA (Fisher Scientific) in PBS containing Tween 20 (Sigma) and Triton X-100 (Sigma) for 30 min and incubated at room temperature under shaking conditions for 2 hr in PBS containing the following anti-mouse primary antibodies: SOCS3 (1:200; Santa Cruz Biotech, Santa Cruz, CA), phospho-CREB (1:600; Cell Signaling, Beverly, MA), total CREB (1:600; Cell Signaling), GFAP, (1:100; Santa Cruz), and IBA1 (1:500; Cedarlane Laboratories, Burlington, NC). After four 15 min washes in filtered PBS, slides were incubated with Cy2 and Cy5-labeled secondary antibodies (all 1:200; Jackson ImmunoResearch, West Grove, PA) for 1 hr under shaking conditions. Following four 15 minute washes with filtered PBS, cells were incubated for 4–5 min with 4′,6-diamidino-2-phenylindole (DAPI, 1:10,000; Sigma). The samples were run in an ethanol and Xylene (Fisher) gradient, mounted and visualized under BX-41 Olympus fluorescence microscope (21 (link)–23 (link)).
Chakrabarti S., Jana M., Roy A, & Pahan K. (2018). Upregulation of suppressor of cytokine signaling 3 in microglia by cinnamic acid. Current Alzheimer research, 15(10), 894-904.
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