Phospho gsk3α β

Manufactured by Cell Signaling Technology

Sourced in United States, Japan

Phospho-GSK3α/β is a lab equipment product that detects the phosphorylation state of glycogen synthase kinase 3 alpha and beta proteins. It is used to measure the activation or inactivation of these enzymes, which play crucial roles in various cellular processes.

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GSK3α/β (45 citations) GSK3α (24 citations) Phospho-GSK3α/β (Ser21/9) (14 citations) Anti-phospho-GSK3α/β (Ser21/9) (10 citations) Phospho-GSK3α (6 citations) Phospho-GSK3α/β (Ser21/Ser9) (2 citations) Anti-phospho (Ser 21/9) GSK3α/β (2 citations)

16 protocols using phospho gsk3α β

Whole Cell Lysate Preparation and Immunoprecipitation

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To prepare whole cell lysates, cells or tissues were lysed in Buffer B as described previously [7 (link)]. For immunoprecipitations, 200 μg whole cell lysate was diluted in IP buffer and immunoprecipitated with 4G10 anti-phosphotyrosine agarose conjugate (05-777, Millipore) as described previously [9 (link)]. Whole cell lysates and immunoprecipitates were processed by WES to separate and visualize cellular proteins according to a standard instrument protocol. Primary antibodies used were: Ron β (#sc-374626, 1:25), GAPDH (#sc-25778, 1:300), PCNA (#sc-56, 1:25) from Santa Cruz Biotechnology; pan AKT (#4691, 1:25), phospho-AKT (#9271, 1:25), S6 (#2217, 1:25), phospho-S6 (#4856, 1:25), phospho-eIF4B (#3591, 1:25), phosphor-AKT1(# 9018, 1:25), phosphor-AKT2 (# 8599, 1:25), AKT1(# 2938, 1:25), AKT2 (# 3063, 1:25), phospho-S6K1 (#9234, 1:25), S6K1 (#2708, 1:25), cleaved PARP (#9541, 1:25), phospho-GSK3α/β (#9331, 1:25), GSK3α/β (#5676, 1:25), Met (#8198, 1:25), Ret (#14556, 1:25) and Axl (#8661, 1:25) from Cell Signaling Technology. Secondary antibodies were included in a Wes Master Kit (PS-MK14, ProteinSimple).

Wang L., Wang L., Cybula M., Drumond-Bock A.L., Moxley K.M, & Bieniasz M. (2020). Multi-kinase targeted therapy as a promising treatment strategy for ovarian tumors expressing sfRon receptor. Genes & Cancer, 11(3-4), 106-121.

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Western Blot Analysis of Drosophila Protein

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For western blotting, total protein from 20 heads of 3-day-old flies was isolated from each indicated group and subjected to SDS-gel electrophoresis. Following transfer, membranes were probed with antibodies to Aβ42 (BioLegend, San Diego, CA, USA), actin (Developmental Studies Hybridoma Bank, Iowa city, IA, USA), GSK-3β (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-Drosophila AKT (Ser505), AKT, phospho-GSK-3α/β (Ser21/9), phospho-Dp70S6K (Thr398), phospho-p38 (Thr180/Tyr182), phospho-ERK (Thr202/Tyr204), ERK, phospho-SAPK/JNK (Thr183/Tyr185) or JNK (Cell Signaling Technology, Beverly, MA, USA). Western blot analyses were conducted using standard procedures with horseradish peroxidase-conjugated secondary antibodies.

Hwang S., Jeong H., Hong E.H., Joo H.M., Cho K.S, & Nam S.Y. (2019). Low-dose ionizing radiation alleviates Aβ42-induced cell death via regulating AKT and p38 pathways in Drosophila Alzheimer's disease models. Biology Open, 8(2), bio036657.

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Multimodal Mitochondrial Function Analysis

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Primary antibodies used: Akt1 (2938), Akt2 (5239), Akt1/2 (9272), GSK-3α/β (5676), LC3 (4108S), Lamin A/C (2032), NFR1 (12381), PGC-1α (2178), phospho-p38 MAPK (9215), phospho-Akt1 (9018), phospho-Akt2 (8599), phospho-DRP1 (Ser616) (4494), phospho-GSK-3α/β (9331), Parkin (2132S), Tfam (7495S), VDAC (4866) (Cell Signaling); β-actin (A5441) (Sigma); PINK1 (ab23707), Mfn-2 (ab124773) (Abcam); Drp1 (611113) (BD Biosciences); phospho-PGC-1α (S571) (AF6650) (R&D Systems); p38 (sc-7972) and Tom20 (sc-11415) (Santa Cruz); SPC (AB3786) (Millipore).

Larson‐Casey J.L., Gu L., Davis D., Cai G., Ding Q., He C, & Carter A.B. (2021). Post‐translational regulation of PGC‐1α modulates fibrotic repair. The FASEB Journal, 35(6), e21675.

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Comprehensive Western Blot and Immunofluorescence Antibodies

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The following antibodies were used in Western blotting and immunofluorescence:
phospho-GSK3α/β (CST 8566), phospho-β-Catenin (Ser33/37/Thr41) (CST9561), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (CST 8814), total β-Catenin (CST 9587), phospho-cyclin D1 (CST 3300), PPARγ (CST 2443), p27 (CST 2552), Ki-67 (CST 9129), phospho-c-Jun (CST 9164), c-Myc (CST 13987), Cytochrome c (CST 4280),GFAP (CST 3670), vimentin (CST 5741), NF-kB (CST 8242), c-Fos (CST 2250) from Cell Signaling Technology (Danvers, MA, US); Lamin B1 (10H34L18), Cyclin D1 (MA5-14512), Bcl-2 (13-8800), phospho-CREB (MA1-083), Ki-67 (MA5-14520) from Invitrogen, Thermo Fisher Scientific, and Phospho-JNK (sc-6254) from Santa Cruz Biotechnology (Dallas, TX, USA).

Ratti S., Rusciano I., Mongiorgi S., Neri I., Cappellini A., Cortelli P., Suh P.G., McCubrey J.A., Manzoli L., Cocco L, & Ramazzotti G. (2021). Lamin B1 Accumulation’s Effects on Autosomal Dominant Leukodystrophy (ADLD): Induction of Reactivity in the Astrocytes. Cells, 10(10), 2566.

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Mitochondrial Dynamics and Signaling Pathways

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Primary antibodies used: Akt1 (2938), Akt2 (5239), Akt1/2 (9272), GSK‐3α/β (5676), LC3 (4108S), Lamin A/C (2032), NFR1 (12381), PGC‐1α (2178), phospho‐p38 MAPK (9215), phospho‐Akt1 (9018), phospho‐Akt2 (8599), phospho‐DRP1 (Ser616) (4494), phospho‐GSK‐3α/β (9331), Parkin (2132S), Tfam (7495S), VDAC (4866) (Cell Signaling); β‐actin (A5441) (Sigma); PINK1 (ab23707), Mfn‐2 (ab124773) (Abcam); Drp1 (611113) (BD Biosciences); phospho‐PGC‐1α (S571) (AF6650) (R&D Systems); p38 (sc‐7972) and Tom20 (sc‐11415) (Santa Cruz); SPC (AB3786) (Millipore).

Larson‐Casey J.L., Gu L., Davis D., Cai G., Ding Q., He C, & Carter A.B. (2021). Post‐translational regulation of PGC‐1α modulates fibrotic repair. The FASEB Journal, 35(6), e21675.

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Immunofluorescence and Western Blotting Assays

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Antibodies raised against Hemagglutinin (Roche Cat# 3F10, RRID:AB_2314622 or Cell Signaling Technology Cat# 3724, RRID:AB_1549585), GRA1 (provided by J.-F- Dubremetz, UMR 5235 Centre National de la Recherche Scientifique, Montpellier, France), Toxofilin (provided by I. Tardieux, INSERM 1209, Grenoble, France), MIC2 (provided by D.Sibley, Washington University School of Medicine, St. Louis, MO), β-catenin (BD Biosciences Cat# 610153, RRID:AB_397554), GSK3β (Cell Signaling Technology Cat# 12456, RRID:AB_2636978), PP2A-B56 (#MABS270, Millipore), PP2A65 RA (Cell Signaling Technology Cat# 2039S, RRID:AB_10695607), PP2Ac (#2038, Cell signaling), Phospho-GSK3α/β (Cell Signaling Technology Cat# 8566S, RRID:AB_10860069), TBP (Abcam Cat# ab62126, RRID:AB_2287049), and TgQRS (van Rooyen et al., 2014 (link)) were used in the immunofluorescence assay and/or Western blotting. Immunofluorescence secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). Western blotting secondary antibodies conjugated to alkaline phosphatase was purchased from Promega. The inhibitors CHIR 99021(#252917-06-9) and BIO (#66746362–9) were purchased from R and D systems. Recombinant bacterial lipopolysaccharide (LPS) from Escherichia coli O26:B6 (Sigma-Aldrich) was used to stimulate the BMDMs.

He H., Brenier-Pinchart M.P., Braun L., Kraut A., Touquet B., Couté Y., Tardieux I., Hakimi M.A, & Bougdour A. (2018). Characterization of a Toxoplasma effector uncovers an alternative GSK3/β-catenin-regulatory pathway of inflammation. eLife, 7, e39887.

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GSK3β Inhibition by Metal Complexes

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Example 25

In order to clearly show that the metal complexes described inhibit GSK3β two sets of cell samples M17 (human) or N2a (murine) neuroblastoma cells were plated at a passaging ratio of 1:4 from a 90% confluent flask of cells and grown until 80-90% confluent. Medium was replaced with fresh serum-free OptiMem and cells were exposed to 1 or 100 nM CuGTSM (from 10 mM stock in DMSO) for 18 hr (overnight). Medium was removed and cells extracted into Phosphosafe (Invitrogen) extraction buffer and frozen at −80° C. Western blots were performed on cell lysates for total GSK3β and phospho-GSK3α/β (Cell Signaling Technology). The blots revealed that GSK3α/β was robustly phosphorylated by treatment with CuGTSM compared to control cells treated with vehicle (DMSO) alone. The phosphorylation of GSK3α/β induces inhibition of GSK3 activity. As GSK3 is known to induce phosphorylation of tau (an important pathological marker in AD), the inhibition of GSK3 activity by CuGTSM would be expected to inhibit tau phosphorylation. The results are shown in FIG. 15.

US10159679B2. Metal delivery agents and therapeutic uses of the same (2018-12-25). The University of Melbourne [AU]. Inventors: Kevin J. Barnham [AU], Paul S. Donnelly [AU], Anthony R. White [AU].

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Protein Expression Analysis in Keratinocytes

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Total protein was extracted from keratinocytes in Leeds lysis buffer [56 (link)] and resolved by SDS-PAGE (10-15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences) and probed with antibodies specific for phosphorylated FRS2α (3861, Cell Signaling Technology), cyclin B1 (H-433, Santa Cruz Biotechnology), involucrin (SY5, Santa Cruz Biotechnology), HPV18 E6 (G-7, Santa Cruz Biotechnology), HPV18 E7 (8E2, Abcam (ab100953), AKT (9272, Cell Signaling Technology), phospho-AKT Ser473 (D9E, Cell Signaling Technology), HA (HA-7, Sigma H9658), cytokeratin 1 (Poly19056, Covance), phospho-ERK1/2 (43705, Cell Signalling Technology), GAPDH (G-9, Santa Cruz Biotechnology), phospho-GSK3α/β (9336, Cell Signalling Technology) and EGFR (R-1, Santa Cruz Biotechnology). Immunoblots were visualized with species-specific HRP conjugated secondary antibodies (Sigma) and ECL (Thermo/Pierce).

Wasson C.W., Morgan E.L., Müller M., Ross R.L., Hartley M., Roberts S, & Macdonald A. (2017). Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle. Oncotarget, 8(61), 103581-103600.

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HOXA9 Regulation of Akt, β-Catenin, and FAK

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Antibodies and reagents were as follows: PTEN (Cell Signaling), HOXA9 for immunofluorescence (a gift from T. Nakamura, Japanese Foundation for Cancer Research, Tokyo, Japan), HOXA9 for western blotting (Santa Cruz), phospho-Akt^Ser473 (Cell Signaling), β-actin (Sigma), Gapdh (Cell Signaling), MYC (Abcam), active b-catenin (Cell Signaling), total β-catenin (Cell Signaling), phospho-GSK3α/β (Cell Signaling), phospho-FAK^Tyr397 (Invitrogen), phospho-Histone H3 (Cell Signaling), secondary AlexaFluor goat anti-mouse and anti-rabbit (Invitrogen) and Matrigel recombinant basement membrane (BD Biosciences). Inhibitors include MYC inhibitor 10058-F4 (10μM, Calbiochem) and FAK Inhibitor 14 (1μM, Tocris).

Mouw J.K., Yui Y., Damiano L., Bainer R.O., Lakins J.N., Acerbi I., Ou G., Wijekoon A.C., Levental K.R., Gilbert P.M., Chen Y.Y, & Weaver V.M. (2014). Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression. Nature medicine, 20(4), 360-367.

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Evaluating Nitric Oxide Signaling Pathways

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DETA NONOate (#82120), 1400W (#80200), and L-NIL (#80300) were purchased from Cayman Chemical (Ann Arbor, MI). Sulfanilamide (#S9251), Sodium Nitrite (#S2252), N-(1-Naphthyl) ethylenediamine dihydrochloride (#22,248-8), and sodium ascorbate (#A7631) were purchased from Sigma-Aldrich (Saint Louis, MO). N-Ethylmaleimide (NEM) (#23030), IP Lysis Buffer (#87787), Protease and Phosphatase Inhibitor Cocktail (#78440), protein quantification assay kit (#A53226) Streptavidin Magnetic beads (#65602), N-[6-(biotinamido) hexyl]-3′-(2′-pyridyldithio)-propionamide (HPDP-Biotin) (#21341) and Protein A Agarose Beads (#20333) were purchased from Thermo Fisher Scientific (Waltham, MA.). LY294002 (#S1105) and Wortmannin (#2758) were purchased from Selleck Chemicals (Houston, TX). All of the cell culture media was purchased from Corning (Corning, NY). All antibodies, including phospho-AKT Ser 473 (pAKTS473)(#4060), AKT (#9272), PTEN (#9559), GSK-3α/β (#5676), phospho-GSK-3α/β (#8566), and PTEN (#9788), were purchased from Cell Signaling Technology (Beverly, CA) unless stated otherwise. We purchased iNOS antibody (#SC-651) from Santa Cruz Biotechnology (Santa Cruz, CA).

Ding Z., Ogata D., Roszik J., Qin Y., Kim S.H., Tetzlaff M.T., Lazar A.J., Davies M.A., Ekmekcioglu S, & Grimm E.A. (2021). iNOS Associates With Poor Survival in Melanoma: A Role for Nitric Oxide in the PI3K-AKT Pathway Stimulation and PTEN S-Nitrosylation. Frontiers in Oncology, 11, 631766.

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