Phospho gsk3α β ser21 9

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-GSK3α/β (Ser21/9) is a lab equipment product that detects the phosphorylation of glycogen synthase kinase 3 (GSK3) at serine 21 and serine 9 residues. It is used to monitor the activation state of GSK3 in cellular samples.

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Lab products found in correlation

Phospho akt ser473, by Cell Signaling Technology (3 mentions) Gsk3α β, by Cell Signaling Technology (3 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Phospho p38 mapk, by Cell Signaling Technology (2 mentions) Ne per nuclear and cytoplasmic extraction reagent kit, by Thermo Fisher Scientific (2 mentions) Albumin, by Abcam (1 mentions) Phosphor tau, by Thermo Fisher Scientific (1 mentions) Tau 46 sc 32274, by Santa Cruz Biotechnology (1 mentions) Phosphor ser thr pka substrate, by Cell Signaling Technology (1 mentions) Gsk3β, by BD (1 mentions) Escherichia coli lps, by Merck Group (1 mentions) Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions) β actin, by Merck Group (1 mentions) Sds polyacrylamide gel, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase pod linked anti rabbit secondary antibody, by GE Healthcare (1 mentions) Anti β actin, by Merck Group (1 mentions) Quantity one software, by Bio-Rad (1 mentions) Phospho p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions) Chemidoc mp analyzer, by Bio-Rad (1 mentions) Anti mkp 1, by Santa Cruz Biotechnology (1 mentions)

14 protocols using phospho gsk3α β ser21 9

Analyzing Alcoholic Liver Disease Proteins

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Frozen liver samples from a previously published study of chronic alcoholic liver disease were also analyzed (14 (link)). Nuclear and cytosolic protein fractions were isolated from TEN control and EtOH-treated rat livers using NE-PER Nuclear and Cytoplasmic Extraction reagent kit (Fisher Scientific, Pittsburg, PA). Blotted proteins were incubated with the anti-active β-catenin antibody, or with a polyclonal antibody recognizing the phosphorylated form of GSK3β, (Phospho-GSK-3α/β (Ser21/9), Cell Signaling Technology, Beverly, MA). Expression of the pro- and active-forms of matrix metalloproteinase-7 (MMP7) were determined in blotted total lysate fractions using a polyclonal anti-MMP7 antibody (Sigma-Aldrich, St. Louis, MO). Primary antibodies were diluted 1:1000 and incubated overnight at 4°C. Secondary antibodies were diluted (1:10,000 to 1:50,000) and incubated at room temperature before chemiluminescense detection. Protein bands were quantified using a densitometer and band densities were corrected for total protein loaded by staining with 0.1% amido black.

Mercer K.E., Hennings L., Sharma N., Lai K., Cleves M.A., Wynne R.A., Badger T.M, & Ronis M.J. (2014). Alcohol consumption promotes diethylnitrosamine-induced hepatocarcinogenesis in male mice through activation of the Wnt/β-catenin signaling pathway. Cancer prevention research (Philadelphia, Pa.), 7(7), 675-685.

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Antibody Characterization for Tau Dysregulation

Cited 1 time

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Protein lysates were prepared as described previously [31 (link)]. Western blot analysis was performed with primary antibodies for GFP (sc-9996, 1:2000), Tau 46 (sc-32274, 1:500), and β-actin (sc-47778, 1:1000) were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA).
Phospho-GSK-3α/β (Ser21/9, 9331s, 1:1000), Phosphor-(Ser/Thr) PKA Substrate (9621s, 1:1000), and Phosphor-Tau (Ser396, PHF13, 9632s, 1:1000) were obtained from Cell Signaling. GSK-3β (Clone 7, 610202, 1:1000) and Drp1 (Clone 8/DLP1, 611112, 1:1000) were obtained from BD Transduction Laboratories. Tau 5 (QF215086, 1:1000), PhosphorTau (Thr205, 44-738G, 1:2000), Phosphor-Tau (Thr214, 44-742G, 1:2000), Phosphor-Tau (Thr231, 44-746G, 1:2000) and (Thr262, 44-750G, 1:2000) were obtained from Thermo Scientific. Phosphor-Tau (Ser409, AB9662, 1:2000) was obtained from Merck Millipore. Albumin (ab106582, 1:1000) was obtained from Abcam. HA monoclonal (H9658, 1:1000) was obtained from Sigma-Aldrich. PKA RII Subunits (06-411, 1:1000) was obtained from EMD Millipore.

Ko H.J., Chiou S.J., Wong Y.H., Wang Y.H., Lai Y.L., Chou C.H., Wang C., Loh J.K., Lieu A.S., Cheng J.T., Lin Y.T., Lu P.J., Fann M.J., Huang C.Y, & Hong Y.R. (2019). GSKIP-Mediated Anchoring Increases Phosphorylation of Tau by PKA but Not by GSK3beta via cAMP/PKA/GSKIP/GSK3/Tau Axis Signaling in Cerebrospinal Fluid and iPS Cells in Alzheimer Disease. Journal of Clinical Medicine, 8(10), 1751.

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Generation and Use of Genetically Modified Mice

Cited 1 time

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Mice lacking p38γ, p38δ and p38γ/δ have been described (Sabio et al., 2005 (link); Risco et al., 2012 (link)). All strains were backcrossed onto the C57BL/6 strain for at least nine generations. B cell-specific p38γ/δ deficient (CD19-Cre^KI/+p38γ/δ^f/f) mice were generated by breeding p38γ/δ^f/f to CD19-Cre^KI/+ mice (Rickert et al., 1997 (link); Alsina-Beauchamp et al., 2018 (link)). Male and female 15–20 weeks old mice were used. Mice were housed in specific pathogen-free conditions in accordance with European Union regulations; work was approved by local CNB-CSIC ethical review and by the Bioethics Committee of the Community of Madrid PROEX316/15. Escherichia coli LPS was purchased from Sigma. Anti-p38α and TPL2 were from Santa Cruz. Antibodies to ERK1/2 and phospho-ERK1/2 (Thr202/Tyr204), Akt and phospho-Akt (Ser473, Thr408), phospho-NFκB1/p105 (Ser933; P-p105), phospho-p38MAPK (Thr180/Tyr182; this antibody recognises all four phosphorylated-p38 isoforms), IκBα and phospho-GSK3α/β (Ser21/9) were from Cell Signaling Technologies; anti-phospho-JNK1/2 (Thr183/Tyr185) was from Biosource. Anti-p38γ and -p38δ antibodies were raised and purified as described (Cuenda et al., 1997 (link)).

Barrio L., Román-García S., Díaz-Mora E., Risco A., Jiménez-Saiz R., Carrasco Y.R, & Cuenda A. (2020). B Cell Development and T-Dependent Antibody Response Are Regulated by p38γ and p38δ. Frontiers in Cell and Developmental Biology, 8, 189.

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Phosphoprotein Analysis of IP6 Treatment

Cited 1 time

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Western blot analysis was performed as described previously [15 (link)]. MG63.3 cells were treated with IP6 (0 or 5 mM) for 6 hours then lysed in SDS gel loading buffer. Lysates from equal number of cells were loaded on 4-20% SDS-PAGE gels. After electrophoresis, proteins were transferred to a nitrocellulose membrane and probed with primary antibodies against phospho-Akt (Ser473), phospho-GSK3α/β (Ser21/9) and phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, Inc. Boston, MA). β-Actin (Sigma, St. Louis, MO) was used as a loading control.

Ren L., Hong E.S., Mendoza A., Issaq S., Hoang C.T., Lizardo M., LeBlanc A, & Khanna C. (2017). Metabolomics uncovers a link between inositol metabolism and osteosarcoma metastasis. Oncotarget, 8(24), 38541-38553.

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Western Blot Analysis of GSK3α/β and HO-1

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Samples (30 μg proteins) were separated on 12% SDS polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and electroblotted onto 0.2 μm nitrocellulose membranes. Membranes were incubated overnight at 4°C with primary antibody recognizing Phospho-GSK3α/β (Ser21/9) (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA) or HO-1 (1:1000; Enzo Life Sciences Inc., Lausanne, Switzerland). Membranes were washed with TBS-T (TBS +0.05% Tween20), and then incubated with a horseradish peroxidase (POD) linked anti-rabbit secondary antibody (1:2000; GE Healthcare, Piscataway, NJ, USA). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Pierce, Rockford, IL, USA). The same membranes were stripped and reprobed with total GSK3α/β (1:1000; Cell Signaling Technology Inc.) or anti-β-actin (1:1000; Sigma-Aldrich, Saint Louis, Missouri, USA). Data were analyzed by densitometry, using Quantity One software (Bio-Rad). Values were normalized and expressed as fold increase of densitometry compared to the sham group.

Morroni F., Sita G., Graziosi A., Turrini E., Fimognari C., Tarozzi A, & Hrelia P. (2018). Neuroprotective Effect of Caffeic Acid Phenethyl Ester in A Mouse Model of Alzheimer’s Disease Involves Nrf2/HO-1 Pathway. Aging and Disease, 9(4), 605-622.

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Quantification of Hepatic Protein Fractions

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Nuclear and cytosolic protein fractions were isolated from TEN control and EtOH-treated rat livers (Ronis et al., 2011) using NE-PER Nuclear and Cytoplasmic Extraction reagent kit (Thermo Fisher Scientific) as per manufacturer’s instructions. Proteins (30 μg) were separated by SDS-polyacrylamide gel electrophoresis using standard methods. Blotted cytosolic proteins were incubated with either an anti-active β-catenin antibody used in immunohistochemistry, or with a polyclonal antibody recognizing the phospohorylated form of GSK3β, (Phospho-GSK-3α/β (Ser21/9), Cell Signaling Technology, Beverly, MA). Total nuclear β-catenin expression was determined using standard procedures and the anti-active β-catenin antibody previously described [14 ]. Protein bands were quantified using a densitometer and band densities were corrected for total protein loaded by staining with 0.1% amido black.

Mercer K.E., Hennings L, & Ronis M.J. (2015). Alcohol consumption, Wnt/β-catenin signaling and hepatocarcinogensis. Advances in experimental medicine and biology, 815, 185-195.

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Western Blot Analysis of Cellular Signaling

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Western blots were performed on WT and AT cell protein extracts, essentially as described in [25 (link)]. After 24-h treatment, cells were collected, washed, and lysed in denaturing lysis buffer for total protein extracts. For cytosolic/nuclear protein extracts, cells were subjected to hypertonic lysis to obtain the cytosolic fraction and subsequent denaturing lysis on the nuclear pellet. Protein extracts were quantified, loaded onto the SDS-PAGE, transferred onto the nitrocellulose membrane and then subjected to immunoblotting. The following antibodies from Cell Signalling Technologies were used in the immunoblotting analyses: Phospho-AKT (Ser473), AKT, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), p44/42 MAPK (ERK1/2), Phospho-GSK-3α/β (Ser21/9), GSK-3α/β, Fyn, LAMIN A/C, p38 MAPK, Phospho-p38 MAPK (Thr180/Tyr182) and KEAP1. Anti-MKP1, NRF2, HPRT1 and β-ACTIN were obtained from Santa Cruz Biotechnology as reported in [25 (link)]. Imaging and quantification were performed using a ChemiDoc MP Analyzer and image lab software (Bio-rad).

Biagiotti S., Bianchi M., Rossi L., Chessa L, & Magnani M. (2019). Activation of NRF2 by dexamethasone in ataxia telangiectasia cells involves KEAP1 inhibition but not the inhibition of p38. PLoS ONE, 14(5), e0216668.

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Phosphorylation Signaling Antibodies

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Anti-phospho-AMPKα Thr172 (#2535), phospho-TAK1 Thr184 (#4537), phospho-GSK-3α/β Ser21/9 (#9331), and phospho-Akt Ser473 (#9271) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-phospho-p70S6K Thr412 (#07–018) and phospho-FOXO Thr32 (#07–694) were purchased from Millipore (Billerica, MA, USA). Anti-phospho-ERK (#M8159) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-phospho-JNK (#44-682G) was purchased from Biosource (Carlsbad, CA, USA). Anti-phospho-p38 MAPK Thr180/Tyr182 (#10009177) was purchased from Cayman Chemical. Anti-GAPDH (#ab36840) was purchased from Abcam (Cambridge, MA, USA). Goat anti-rabbit secondary antibody (#ALI4404) was purchased from Biosource. Rabbit anti-mouse secondary antibody (#A9044) was purchased from Sigma-Aldrich. Morpholinos were purchased from Gene Tools, LLC (Philomath, OR, USA).

Glennon E.K., Torrevillas B.K., Morrissey S.F., Ejercito J.M, & Luckhart S. (2017). Abscisic acid induces a transient shift in signaling that enhances NF-κB-mediated parasite killing in the midgut of Anopheles stephensi without reducing lifespan or fecundity. Parasites & Vectors, 10, 333.

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Tissue Analysis of Lung Tumors

Cited 3 times

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Tissue isolation, fixation and staining procedures were performed as previously described (Momcilovic et al., 2017 (link); Momcilovic et al., 2015 (link); Shackelford et al., 2013 (link)). Briefly, lungs or tumors were fixed overnight in 10% buffered formalin, and then transferred to 70% ethanol. Tissues were processed and embedded by TPCL at UCLA. The following antibodies were used: phospho-4EBP1 (Thr37/46) (Cell Signaling Technology, #2855 1:800), anti-CK5 (EP1601Y) (abcam, ab52635 1:100), anti-TTF1 (8G7G3/1) (Dako, 1:1000), anti-Ki67 (SP6) (Thermo Scientific, RM-9106-S0 1:200), phospho-S6 (Cell Signaling Technology, #4585 1:400), phospho-GSK3α/β (Ser21/9) (Cell Signaling Technology, #9331 1:50), c-Jun (Cell Signaling Technology, #9165 1:800), phospho-c-Jun (S73) (D47G9) (Cell Signaling Technology, #3270 1:200), anti-SLC1A5 (Sigma, HPA035240, 1:400), anti-GLUT1 (Alpha Diagnostic, GT11A, 1:400), anti-Myc (Y69) (abcam, ab32072, 1:100). Slides were scanned onto a ScanScope AT (Aperio Technologies, Inc., Vista, CA). Digital slides were analyzed with QuPath software in order to determine percent positive cells for Ki67, GLUT1 and p4EBP1 stains.

Momcilovic M., Bailey S.T., Lee J.T., Fishbein M.C., Braas D., Go J., Graeber T.G., Parlati F., Demo S., Li R., Walser T.C., Gricowski M., Shuman R., Ibarra J., Fridman D., Phelps M.E., Badran K., John M.S., Bernthal N.M., Federman N., Yanagawa J., Dubinett S.M., Sadeghi S., Christofk H.R, & Shackelford D.B. (2018). The GSK3 signaling axis regulates adaptive glutamine metabolism in lung squamous cell carcinoma. Cancer cell, 33(5), 905-921.e5.

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Protein Extraction from Small Intestine Tissue

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To prepare protein lysates, snap-frozen small intestine samples were homogenized on ice for 1 min in ice-cold RIPA lysis buffer (150 mM NaCl, 50 mM Tris pH 7.0, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100 and 0.5% NP40) supplemented with protease and phosphatase inhibitor cocktail (Calbiochem 539134 and Sigma P0044 respectively). Samples were centrifuged at 14,000 x g for 10 min at 4°C to remove insoluble debris and supernatant collected. The protein concentration of supernatants was determined by the Bradford's method (Pierce). Primary antibodies used were: β-Catenin (BD Transduction 610153), phospho-GSK3α/β (Ser21/9) (Cell Signalling 9331), GSK3α/β (Cell Signalling 5676), ERK2 (Santa Cruz sc-1647) GAPDH (Millipore, MAB374), cleaved Caspase 3 (Cell Signalling 7661) and cleaved PARP (Enzo BML-SA249).

Hey F., Giblett S., Forrest S., Herbert C, & Pritchard C. (2016). Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine. PLoS ONE, 11(6), e0156877.

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