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Phospho crebs133

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Phospho-CREB (Ser133) is a monoclonal antibody that recognizes CREB protein phosphorylated at serine 133. CREB is a transcription factor that plays a role in cellular responses to various stimuli.

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Lab products found in correlation

Cox 4, by Cell Signaling Technology (3 mentions) Protease inhibitor cocktail, by Roche (3 mentions) Phospho pka substrates t, by Cell Signaling Technology (3 mentions) β actin, by Cell Signaling Technology (2 mentions) Phospho akt s473, by Cell Signaling Technology (2 mentions) Phospho p38t180 y182, by Cell Signaling Technology (2 mentions) Total oxphos rodent antibody cocktail, by Abcam (1 mentions) α tubulin, by Cell Signaling Technology (1 mentions) Hsp90, by Cell Signaling Technology (1 mentions) Caspase 9, by Cell Signaling Technology (1 mentions) Phospho paxillin y118, by Cell Signaling Technology (1 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Phospho gsk3β s9, by Cell Signaling Technology (1 mentions) Phospho chk1 s345, by Cell Signaling Technology (1 mentions) Cellytic mt reagent, by Merck Group (1 mentions) γh2ax, by Cell Signaling Technology (1 mentions) β actin, by Proteintech (1 mentions) β tubulin, by Proteintech (1 mentions) Cyclin b1, by Santa Cruz Biotechnology (1 mentions) Phospho atg14 s29, by Cell Signaling Technology (1 mentions)

8 protocols using phospho crebs133

1

Adipose Tissue Protein Isolation and Western Blot

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Total protein from mouse adipose tissue was prepared in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) supplemented with a protease inhibitor cocktail (Roche) and phosphatase inhibitors (10 mM NaF, 60 mM β-glycerolphosphate, pH 7.5, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate). Proteins were subjected to SDS-PAGE then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were probed with following antibodies: UCP1 (Abcam, ab10983), Total OXPHOS Rodent Antibody Cocktail (Abcam, ab110413), COXIV (Cell Signaling, 4850), VDAC (Cell Signaling, 4661), HSP90 (Cell Signaling, 4874), phospho-PKA substrateS/T (Cell Signaling, 9621), phospho-CREBS133 (Cell Signaling, 9198), CREB (Cell Signaling, 9197), β-actin (Cell Signaling, 8457) and α-tubulin (Cell Signaling, 2144).
Jun H., Ma Y., Chen Y., Gong J., Liu S., Wang J., Knights A.J., Qiao X., Emont M.P., Xu X.Z., Kajimura S, & Wu J. (2020). Adrenergic-independent signaling via CHRNA2 regulates beige fat activation. Developmental cell, 54(1), 106-116.e5.
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2

Western Blot Analysis of Signaling Proteins

Cited 1 time
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Western blot was performed as previously described [23 (link)]. Antibodies of c-myc (product #5605), phospho-paxillin (Y118) (#2541), phospho-CREB (S133) (#9198), CREB (#9197), caspase-3 (#9665), caspase-9 (#9502), cleaved PARP XP (#5625), and β-actin (#4970) were purchased from Cell Signaling Technology.
Justus C.R., Sanderlin E.J., Dong L., Sun T., Chi J.T., Lertpiriyapong K, & Yang L.V. (2017). Contextual tumor suppressor function of T cell death-associated gene 8 (TDAG8) in hematological malignancies. Journal of Translational Medicine, 15, 204.
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3

Profiling Phosphorylation Signaling Pathways

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Protein extracts from SK-BR-3 cells were separated by SDS-PAGE, transferred onto PVDF membranes, and probed with antibodies against phospho-AKT (S473) (Cell Signaling #9271), phospho-p53 (S46) (Cell Signaling #2521), phospho-GSK3β (S9) (Cell Signaling #5558), phospho-p44/42 (ERK1: T202/Y204; ERK2: T185/Y187) (R&D Systems #AF1018), phospho-CREB (S133) (Cell Signaling #9196), and actin (Santa Cruz Biotechnology #sc47778). Proteins were detected with HRP-conjugated secondary antibodies and visualized with the Pierce ECL Western blotting substrate (ThermoScientific) according to the manufacturer's protocol.
Montoya A., Amaya C.N., Belmont A., Diab N., Trevino R., Villanueva G., Rains S., Sanchez L.A., Badri N., Otoukesh S., Khammanivong A., Liss D., Baca S.T., Aguilera R.J., Dickerson E.B., Torabi A., Dwivedi A.K., Abbas A., Chambers K., Bryan B.A, & Nahleh Z. (2016). Use of non-selective β-blockers is associated with decreased tumor proliferative indices in early stage breast cancer. Oncotarget, 8(4), 6446-6460.
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4

Protein Extraction and Western Blot Analysis

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Total protein from fat tissues and differentiated primary preadipocytes was prepared in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) supplemented with a protease inhibitor cocktail (Roche) and phosphatase inhibitors (10 mM NaF, 60 mM β-glycerolphosphate, pH 7.5, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate). Proteins were subjected to SDS-PAGE, and were then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were probed with following antibodies: from Abcam: UCP1 (ab10983), MitoProfile total OXPHOS (ab110413); from Cell Signaling: COXIV (#4850), phospho-PKA substrateS/T (#9621), phospho-CREBS133 (#9198), CREB (#9212), phospho-p38T180/Y182 (#9215), p38 (#9197) α-tubulin (#2144), β-actin (#8457), GAPDH (#5174), and HSP90 (#4874). Phosphorylated CREB and p38 normalized to total CREB and p38, respectively, were quantified using ImageJ software.
Jun H., Yu H., Gong J., Jiang J., Qiao X., Perkey E., Kim D.I., Emont M.P., Zestos A.G., Cho J.S., Liu J., Kennedy R.T., Maillard I., Xu X.Z, & Wu J. (2018). An immune-beige adipocyte communication via nicotinic acetylcholine receptor signaling. Nature medicine, 24(6), 814-822.
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5

Comprehensive Western Blot Analysis

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Cells were lysed on ice with CelLytic MT reagent (Sigma) supplemented with protease and phosphatase inhibitors. Primary antibodies: TSC1 (#6935), TSC2 (#4308), phospho-S6 (#4858), S6 (#2217), p21 (#2947), Lamin B1 (#12586), LC3 A/B (#12741), phospho-ATG14-S29 (#92340), ATG14 (#96752), phospho-CREB-S133 (#9198), CREB (#9197), phospho-CHK1-S345 (#2348), phospho-WEE1-S642 (#4910), WEE1(#13084), PLK1 (#4513), γH2A.X (#9718), phospho-S6K-S371 (#9208), S6K (#2708), phospho-4EBP1-S65 (#9451), 4EBP1 (#9644), phospho-AKT-S473 (#4060), AKT (#4691), p53 (#2527), phospho-CDC2-T15 (#9111), CDC2 (#9116), and phospho-ATR-S428 (#2853) from Cell signaling (all used at a 1: 1000 dilution); β-tubulin (#10094-1-ap, 1: 4000), β-actin (#20536-1-ap, 1: 4000), and GAPDH (#10494-1-ap, 1: 10000) from ProteinTech; ATR (#A300-137A-T, 1: 1000) from Bethyl Lab, and CHK1 (#sc-8408, 1:1000) and Cyclin B1(#sc-245, 1: 1000) from Santa Cruz.
Liu Y., Azizian N.G., Sullivan D.K, & Li Y. (2022). mTOR inhibition attenuates chemosensitivity through the induction of chemotherapy resistant persisters. Nature Communications, 13, 7047.
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6

Reagents and Antibodies for Neuronal Studies

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The CMV-mCherry-dynamitin expression vector was kindly shared by M. Meffert (Johns Hopkins, MD; Shrum et al., 2009 (link)) while the mCherry plasmid was a gift from R.Y. Tsien (UC San Diego, CA). The 4xGFP construct was a gift from W. Hampe (UMC Hamburg-Eppendorf, Hambug; Seibel et al., 2007 (link)). Commercial plasmids include Dendra2 (Evrogen) and CRTC1 (Open Biosystems, Huntsville, AL). Antibodies used in all these experiments include: rabbit polyclonal antibodies against CRTC1 (Bethyl, Montgomery, TX and Proteintech, Chicago, IL), pCRTC1(S151; Bethyl) Dendra2 (Evrogen, Moscow, Russia), TUJ1 (Covance, Princeton, NJ), Dynein heavy chain (Santa Cruz, Dallas, TX), and phosphoCREB-S133 (Cell Signaling); mouse monoclonal antibodies against PSD95 (Thermoscientific, Rockford, IL), synapsin1 (Millipore, Billerica, MA), CamKIIα (Millipore), HA-epitope (Sigma), GAPDH (Fitzgerald, Acton, MA), GFP (Clontech, Mt. View, CA), GAD67 (Millipore), and KPNB1 (ABR, Golden, CO); polyclonal chicken antibody against MAP2 (Phosphosolutions, Aurora, CO) and synaptotagmin (Chemicon, Temecula, CA). All secondary antibodies are conjugated to Alexa dyes (488, 546, 555, 568, and 633; Invitrogen).
Ch'ng T.H., DeSalvo M., Lin P., Vashisht A., Wohlschlegel J.A, & Martin K.C. (2015). Cell biological mechanisms of activity-dependent synapse to nucleus translocation of CRTC1 in neurons. Frontiers in Molecular Neuroscience, 8, 48.
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7

Protein Extraction and Western Blot

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Cells were harvested and disrupted in a buffer containing 50mM HEPES pH 7.5, 150mM NaCl, 1% (v/v) glycerol; 1% (w/v) Triton X-100, 1.5mM MgCl2, 5mM EGTA, protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (Sigma-Aldrich). 10 to 30μg of total protein were resolved by SDS-PAGE and transferred to the nitrocellulose membrane, which was incubated overnight with specific antibodies: vinculin, Grp78, CHOP (GADD153) IDH1 and GSS from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); phospho-(Ser/Thr) PKA Substrate, phospho-CREB S133, total CREB, Beclin-1, LC3, Eif2α, cleaved caspase 3, phospho-Src (Y416) and total Src (36D10) from Cell Signaling Technology Inc. (Danvers, MA, USA); O-Linked N-Acetylglucosamine (Clone RL2) from Thermo Scientific (Waltham, MA, USA).
Palorini R., Votta G., Pirola Y., De Vitto H., De Palma S., Airoldi C., Vasso M., Ricciardiello F., Lombardi P.P., Cirulli C., Rizzi R., Nicotra F., Hiller K., Gelfi C., Alberghina L, & Chiaradonna F. (2016). Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis. PLoS Genetics, 12(3), e1005931.
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8

Protein Extraction and Western Blot Analysis

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Total protein from fat tissues and differentiated primary preadipocytes was prepared in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) supplemented with a protease inhibitor cocktail (Roche) and phosphatase inhibitors (10 mM NaF, 60 mM β-glycerolphosphate, pH 7.5, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate). Proteins were subjected to SDS-PAGE, and were then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were probed with following antibodies: from Abcam: UCP1 (ab10983), MitoProfile total OXPHOS (ab110413); from Cell Signaling: COXIV (#4850), phospho-PKA substrateS/T (#9621), phospho-CREBS133 (#9198), CREB (#9212), phospho-p38T180/Y182 (#9215), p38 (#9197) α-tubulin (#2144), β-actin (#8457), GAPDH (#5174), and HSP90 (#4874). Phosphorylated CREB and p38 normalized to total CREB and p38, respectively, were quantified using ImageJ software.
Jun H., Yu H., Gong J., Jiang J., Qiao X., Perkey E., Kim D.I., Emont M.P., Zestos A.G., Cho J.S., Liu J., Kennedy R.T., Maillard I., Xu X.Z, & Wu J. (2018). An immune-beige adipocyte communication via nicotinic acetylcholine receptor signaling. Nature medicine, 24(6), 814-822.
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