Anti phospho gsk3β

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-phospho-GSK3β is a laboratory research antibody that specifically detects the phosphorylated form of glycogen synthase kinase 3 beta (GSK3β). GSK3β is a serine/threonine protein kinase that plays a key role in numerous cellular processes. The phosphorylation state of GSK3β is an important regulatory mechanism for its activity.

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58 protocols using anti phospho gsk3β

Immunoblotting Analysis of Signaling Proteins

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We used the following antibodies: anti-Akt (No. 9272), anti-phospho-Akt (Ser473; No. 9271), anti-S6K (No. 9202), anti-phospho-S6K (Thr389; No. 9205), anti-S6 (No. 2217), anti-phospho-S6 (Ser235/236; No. 4858), anti-4EBP1 (No. 9644), anti-phospho-4EBP1 (Thr37/46; No. 2855), anti-GSK3β (No. 12456), anti-phospho-GSK3β (Ser9; No. 5558), anti-Ampk (No. 2532), anti-phospho-Ampk (Ser9; No. 2535), anti-mouse IgG (No. 7076), and anti-rabbit IgG (No. 7074) purchased from Cell Signaling Technology (Massachusetts, United States of America). Anti-α-Tubulin (12G10) was purchased from DSHB (Iowa, United States of America). Anti- Laminin α-2 (SC-59854) was purchased from Santa Cruz Biotechnology (Texas, United States of America).

Aoki Y., Ozawa T., Numata O, & Takemasa T. (2019). High-Molecular-Weight Polyphenol-Rich Fraction of Black Tea Does Not Prevent Atrophy by Unloading, But Promotes Soleus Muscle Mass Recovery from Atrophy in Mice. Nutrients, 11(9), 2131.

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Western Blot Analysis of Nuclear and Cytoplasmic Proteins

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Nuclear and cytoplasmic extracts were prepared from liver tissue and cultured cells using a commercially available nuclear extraction kit (Cayman Chemical Co., Ann Arbor, MI, USA). Twenty micrograms of protein was resolved on a 4%-12% Bis-Tris NuPAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot Transfer Stack (Invitrogen). After protein transfer, the following primary antibodies were used: anti-phospho-GSK-3β (#9323; Cell Signaling Technology, Danvers, MA, USA), anti-GSK-3β (#9315; Cell Signaling Technology, Danvers, MA, USA), anti-β-catenin (#9582; Cell Signaling Technology, Danvers, MA, USA), anti-TCF4 (#2569; Cell Signaling Technology), anti-PPARγ (#2443; Cell Signaling Technology), anti-SREBP-1 (sc-366; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-β-actin (#4967; Cell Signaling Technology), and anti-Lamin B1 (ab16048; Abcam, Cambridge, MA, USA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected by chemiluminescence (GE Healthcare, Piscataway, NJ, USA).

Seo M.H., Lee J., Hong S.W., Rhee E.J., Park S.E., Park C.Y., Oh K.W., Park S.W, & Lee W.Y. (2016). Exendin-4 Inhibits Hepatic Lipogenesis by Increasing β-Catenin Signaling. PLoS ONE, 11(12), e0166913.

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Adipocyte Protein Expression Analysis

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Cells were lysed and tissues were homogenized by Polytron homogenizer immediately after dissection in western extraction buffer (150 mmol/L NaCl, 10% glycerol, 1% NP-40, 1 mmol/L EDTA, 20 mmol/L NaF, 30 mmol/L sodium pyrophosphate, 0.5% sodium deoxycholate, 0.05% SDS, 25 mmol/L Tris-HCl: pH 7.4) containing protease inhibitor cocktail (Roche). The lysate was then sonicated, and debris was removed by centrifugation. SDS-PAGE and western blotting were performed and detected with ECL (Thermo Scientific). Antibodies used for western blot analysis were as follows: anti-Adipsin (R&D Systems, catalog# AF5430), anti-adiponectin (Invitrogen, catalog# PA1-054), anti-C3 (Proteintech, catalog# 21337-1-AP), anti-FABP4 (Cell Signaling Technology, catalog# 2120), anti-phospho-GSK-3β (Cell Signaling Technology, catalog# 9336), anti-HSP90 (Proteintech Group, Inc, catalog# 1371-1-AP), and anti-β‐Catenin (Cell Signaling Technology, catalog# 9562).

Aaron N., Kraakman M.J., Zhou Q., Liu Q., Costa S., Yang J., Liu L., Yu L., Wang L., He Y., Fan L., Hirakawa H., Ding L., Lo J., Wang W., Zhao B., Guo E., Sun L., Rosen C.J, & Qiang L. (2021). Adipsin promotes bone marrow adiposity by priming mesenchymal stem cells. eLife, 10, e69209.

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Immunoblot Analysis of INS-1 Cell Lysates

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After treatment, INS-1 cells were harvested and cell lysates were made. After concentration being measured, protein was subjected to SDS-PAGE gel under reducing condition and transferred onto a nitrocellulose membrane. After blocking with 5% milk, the membrane was incubated with anti-β actin, anti-phospho-GSK-3β (Cell signaling), anti-GSK-3β (Cell signaling), anti-SOD (Abcam), and anti-NOX4 antibodies (Abcam) at 4°C overnight. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Jackson Labs). The reaction was visualized using an enhanced chemiluminescence system (Pierce). Immunoblots were analyzed by scanning densitometry and quantified by Quantity One gel Analysis software (Bio-Rad Laboratories).

Guo B., Zhang W., Xu S., Lou J., Wang S, & Men X. (2015). GSK-3β mediates dexamethasone-induced pancreatic β cell apoptosis. Life sciences, 144, 1-7.

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Immunoblot Analysis of Myogenesis Markers

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Immunoblot analysis was performed to detect protein expression of Stat-3, and MyoD and Pax7 (myogenic transcripton factor and satellite cell activation markers, respectively), and phosphorylation of Akt, Glut4, GSK-3β (markers of insulin signaling) and Stat-3 in myotubes. For protein analysis, 40 μg of protein extract per lane was fractionated by Nu-PAGE and blotted onto a nitrocellulose membrane (Invitrogen, Carlsbad, CA). For regular antibodies, the membrane was blocked by 5% nonfat dry milk and 1% nonfat dry milk for phospho antibodies for 30 min. The membranes were stained with anti-phospho Akt, anti-phospho Glut4, anti-phospho Stat-3 (Santa Cruz, Dallas, TX), anti-phospho GSK-3β (Cell Signaling, Danvers, MA), anti-Stat-3, anti-MyoD and anti-Pax7 (Santa Cruz, Dallas, TX), and anti-actin (1:10,000) (Sigma, St. Louis, MO) primary antibodies overnight at 4 °C. The membranes were incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies for 30 min (dilution of 1:5,000) and proteins detected by exposing membranes to Kodak X-Omat films.

Khan M.A., Khan M.F., Kashiwagi S., Kem W.R., Yasuhara S., Kaneki M., Tompkins R.G, & Jeevendra Martyn J.A. (2017). An Alpha7 Nicotinic Acetylcholine Receptor Agonist (GTS-21) Promotes C2C12 Myonuclear Accretion in Association with Release of Interleukin-6 (IL-6) and Improves Survival in Burned Mice. Shock (Augusta, Ga.), 48(2), 227-235.

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Investigating IGF-I Signaling Pathways

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Recombinant human IGF-I was purchased from R&D System (Wiesbaden, Germany). The dual IGF-IR/IR inhibitor OSI-906 was purchased from SelleckBio (USA). Specific PI3K/Akt inhibitor LY294002 was purchased from Sigma (St. Louis, MO), and specific ERK1/2 inhibitor PD98059 was purchased from Promega (Madison, WI). Proteasome inhibitor bortezomib (PS-341) was purchased from Millenium Pharmaceuticals Inc (Cambridge, MA, USA). Anti-E-cadherin, anti-Vimentin, anti-ZEB1, anti-IGF-IR, anti-phospho-IGF-IR (Tyr1131), anti-phospho-GSK-3β, anti-GSK-3β and anti-phospho-P53 (Ser15) were purchased from Cell Signaling Technology (Beverly, MA). Anti-Snail and anti-Twist2 were purchased from Abcam (Cambridge, MA). All the other antibodies were purchased from Santa Cruz Biotechnology (USA).

Li H., Xu L., Li C., Zhao L., Ma Y., Zheng H., Li Z., Zhang Y., Wang R., Liu Y, & Qu X. (2014). Ubiquitin ligase Cbl-b represses IGF-I-induced epithelial mesenchymal transition via ZEB2 and microRNA-200c regulation in gastric cancer cells. Molecular Cancer, 13, 136.

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Epithelial-Mesenchymal Transition Signaling Pathway Analysis

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The following antibodies were applied: Anti-ARHGAP10 (Catalog No. sc-390145, Santa Cruz). Anti-E-Cadherin (Catalog No. ab40772, abcam). Anti-N-Cadherin (Catalog No. ab76011, abcam). Anti-Vimentin (Catalog No. ab92547, abcam). Anti-Snail (Catalog No. ab216347, abcam). Anti-PI3K (Catalog No. 4249, Cell Signaling Technology). Anti-Akt (Catalog No. 4691, Cell Signaling Technology). Anti-phospho-Akt (Catalog No. 4060, Cell Signaling Technology). Anti-GSK3β (Catalog No. ab32391, abcam). Anti-phospho-GSK3β (Catalog No.9323, Cell Signaling Technology). Anti-β-catenin (Catalog No. 8480, Cell Signaling Technology). Anti-GAPDH (Catalog No. ab181602, abcam).

Lin L.L., Yang F., Zhang D.H., Hu C., Yang S, & Chen X.Q. (2021). ARHGAP10 inhibits the epithelial–mesenchymal transition of non-small cell lung cancer by inactivating PI3K/Akt/GSK3β signaling pathway. Cancer Cell International, 21, 320.

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Cholesteatoma Keratinocyte Signaling Pathways

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Cholesteatoma keratinocytes were cultured on 35 mm culture dishes. The cells were grown to 70-80% confluence and then placed in KSFM with control (DMSO), GW0742 (100 nM), or GSK0660 (5 μM). After 24 h of treatment, the cells were washed and isolated using cell lysis buffer (Beyotime Institute of Biotechnology, China) containing protease inhibitors. Equal amounts of total protein were separated on 8% SDS-PAGE and transferred to a PVDF membrane (100 V for 60 min). The membrane was incubated with the primary antibodies overnight at 4°C, followed by the secondary peroxidase-conjugated antibody for 1 h. The bands were visualized by enhanced chemiluminescence and exposure to ECL Hyperfilm (GE Healthcare). The densitometry of bands was quantified with NIH Image 1.63 software. The protein expression was normalized to the amount of beta-actin. The following primary antibodies were used: anti-phospho-PDK1, anti-protein kinase B (AKT), anti-phospho-AKT, anti-phospho-GSK3β, and anti-phospho-PTEN (all from Cell Signaling Technology, Danvers, MA, USA). Antibodies against β-actin, Cyclin D1, and PPAR β/δ were from Genetex, Inc. (Genetex, CA, USA).

Zhang C., Liu Y.W., Chi Z, & Chen B. (2020). Ligand-Activated Peroxisome Proliferator-Activated Receptor β/δ Facilitates Cell Proliferation in Human Cholesteatoma Keratinocytes. PPAR Research, 2020, 8864813.

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Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed as described previously [34 (link)]. The following antibodies were used in this study: anti-AKT (Cell Signaling, 2920S), anti-phospho-ERK(CellSignaling,4370S), anti-phospho-AKT(Ser473) (Epitomics,2118-1), anti-GAPDH(Santa Cruz Biotechnology, SC-32233), PARP(Santa Cruz Biotechnology, SC-8007), anti-caspase-3(Cell Signaling, 9665S), anti-actin(Sigma, A4700), anti-cytochrome C(Santa Cruz Biotechnology, SC-13156), anti-Tom20(Santa Cruz Biotechnology, SC-136211), anti-Mcl-1(Santa Cruz Biotechnology, SC-819), anti-GFP(Sigma, G1544), anti-ERK(Cell Signaling, 4695S), anti-TNFAIP1(proteintech, 60327), anti-LIN28(proteintech,11724), anti-GBP1(proteintech, 15303), anti-Bcl-2(proteintech, 12789), anti-Bxl-xl(proteintech, 26967), anti-GSK3α(Cell Signaling, 9338), anti-phospho-GSK3α(ser21)(Boster, P03152), anti-GSK3β(Cell Signaling, 12456), anti-phospho-GSK3β(ser9)( Cell Signaling, 5558).

Shuang W., Hou L., Zhu Y., Li Q, & Hu W. (2017). Mcl-1 stabilization confers resistance to taxol in human gastric cancer. Oncotarget, 8(47), 82981-82990.

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Investigating PPAR-α Signaling Pathways

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BZA was acquired from Sigma (B7273, purity > 98%). Phenylephrine (PE, P1240000) was obtained from Sigma-Aldrich. Anti-PPAR-α (sc-9000) and anti-PCNA (sc-7907) were purchased from Santa Cruz Biotechnology. The following first antibodies were purchased from Cell Signaling Technology: anti-AKT (#4691), anti-phospho-AKT (#4060), anti-GSK3β (#9315), anti-phospho-GSK3β (#9323P), anti-ERK (#4695), anti-phospho-ERK (#4370P), anti-P38 (#9212P), anti-phospho-P38 (#4511P), anti-AMPKα (#2603P), and anti-phospho-AMPKα (#2535). Anti-GAPDH (#ab8245), anticalcineurin (CaN) (#ab90540), and anti-NFAT1 (#ab2722) were obtained from ABCAM. Anti-α-actinin was acquired from Millipore. The secondary antibodies were purchased from LI-COR Biosciences. The PPAR-α antagonist (GW6471, G5045), PPAR-β/δ antagonist (GSK0660, G5797), and PPAR-γ antagonist (GW9662, M6191) were all purchased from Sigma-Aldrich. All other chemicals were of analytical grade.

Xu S.C., Ma Z.G., Wei W.Y., Yuan Y.P, & Tang Q.Z. (2017). Bezafibrate Attenuates Pressure Overload-Induced Cardiac Hypertrophy and Fibrosis. PPAR Research, 2017, 5789714.

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