Phospho gsk3β

Manufactured by Cell Signaling Technology

Sourced in United States, China, United Kingdom, Canada

Phospho-GSK3β is a primary antibody that recognizes the phosphorylated form of glycogen synthase kinase 3 beta (GSK3β) at serine 9. GSK3β is a multifunctional serine/threonine protein kinase involved in the regulation of various cellular processes.

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Lab products found in correlation

106 protocols using phospho gsk3β

Dexamethasone and CNP-22 Regulate MAPK Signaling

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ATDC5 cells were plated in 6-well tissue culture plates and differentiated for 14 days as described above. Differentiated ATDC5 cells were incubated in insulin-free medium for 2 days. Then after the incubation with vehicle or 10⁻⁶ M DEX^{51 (link)} for 30 minutes, vehicle or 10⁻⁶ M CNP-22^{20 (link)} was added into the medium for 30 minutes. Total protein was extracted from ATDC5 cells by RIPA buffer containing SDS solution and a protease inhibitor cocktail (No. 08714-04, Nacalai), which was supplemented with phosphatase inhibitors (No. 07574-61, Nacalai). Western blotting was performed using the following primary antibodies: Erk 1/2 (No. 4695S, Cell Signaling Technology), Phospho-Erk 1/2 (No. 4376S, Cell Signaling Technology), p38 (No. 9212S, Cell Signaling Technology), Phospho-p38 (No. 9211S, Cell Signaling Technology; RRID), GSK3β (No.9315S, Cell Signaling Technology), and Phospho-GSK3β (No. 9323 S, Cell Signaling Technology).

Ueda Y., Yasoda A., Hirota K., Yamauchi I., Yamashita T., Kanai Y., Sakane Y., Fujii T, & Inagaki N. (2019). Exogenous C-type natriuretic peptide therapy for impaired skeletal growth in a murine model of glucocorticoid treatment. Scientific Reports, 9, 8547.

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Signaling Pathway Activation in Macrophages

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WT and Dgkζ-/- BMMs treated with 100 ng/ml LPS, 100ng/ml IFNγ or 50 ng/ml IL-4 for the indicated time points were lysed in RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% sodium deoxycholate) supplemented with protease/phosphatase inhibitor cocktail (Pierce). The protein lysates were resolved by SDS-PAGE and electro-transferred into nitrocellulose membranes and probed with specific antibodies. The following antibodies were used: phospho-STAT1 (7649, Cell Signaling Technology), STAT1 (9172, Cell Signaling Technology), phospho-STAT3 (9145, Cell Signaling Technology), STAT3 (4904, Cell Signaling Technology), phospho-STAT6 (9361, Cell Signaling Technology), STAT6 (5397, Cell Signaling Technology), phospho-AKT (4060, Cell Signaling Technology), AKT (2966, Cell Signaling Technology), phospho-p38 MAPK (9216, Cell Signaling Technology), p38 MAPK (9219, Cell Signaling Technology), phospho-GSK3β (9336, Cell Signaling Technology), GSK3β (9315, Cell Signaling Technology) and β-Actin (A5441, Sigma).

Mahajan S., Mellins E.D, & Faccio R. (2019). Diacylglycerol kinase ζ regulates macrophage responses in juvenile arthritis and cytokine storm syndrome mouse models. Journal of immunology (Baltimore, Md. : 1950), 204(1), 137-146.

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Western Blot Analysis of L. amazonensis Infection

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Differentiated THP-1 cells were infected with L. amazonensis, and total and nuclear extract proteins were obtained [13 (link)]. Both nuclear (10 µg) and total proteins (30 µg) were subjected to electrophoresis in 10% SDS-polyacrylamide gels. The proteins were transferred to a nitrocellulose membrane (Amersham). Blots were separately incubated with primary antibody against p50 (Millipore—06-886), lamin A/C (Cell Signalling 2032), phospho105 (serine 907) (sc101746), phospho GSK3β (Cell Signalling 9326S), GSK3β (Cell Signalling 931S), phosphoPTEN (Serine 370) (GenScript A00290), Akt (Cell Signalling 9272S) and β-actin (Sigma-Aldrich A2228). Antibody anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG (1 : 3000) was used. The membranes were then submitted to three washings with TBST, and proteins were detected by the ECL chemiluminescent detection system (Amersham Biosciences).

Calegari-Silva T.C., Vivarini Á.C., Miqueline M., Dos Santos G.R., Teixeira K.L., Saliba A.M., Nunes de Carvalho S., de Carvalho L, & Lopes U.G. (2015). The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway. Open Biology, 5(9), 150118.

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Western Blot Analysis of Osteogenic Markers

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Cell extracts containing 40 μg of total protein were separated by electrophoresis on sodium dodecyl sulphate polyacrylamide gels, and subsequently transferred to nitrocellulose membranes. After transfer, the membranes were blocked with PBS containing 5% non‐fat milk for 1 hour at room temperature, and then incubated at 4°C overnight with primary antibodies against Total AKT (T‐AKT, Boster, Wuhan, China), Phospho‐AKT (P‐AKT; Cell Signaling Technology), T‐GSK‐3β (Cell Signaling Technology), Phospho‐GSK‐3β (Cell Signaling Technology), β‐catenin (Cell Signaling Technology), Cyclin D1 (Abcam, Cambridge, MA, USA), Runt‐related transcription factor 2 (RUNX2; Boster), Bone morphogenic protein‐2 (BMP2; Boster) and Bone sialoprotein (BSP; Boster). Antibody against β‐actin (Sigma‐Aldrich) was used as normalizing control. The membranes were subsequently incubated with the secondary antibodies for 1 hour at room temperature. The results were analysed using an Odyssey 2‐colour infrared laser imaging system (LI‐COR Biosciences, Lincoln, NE, USA). Relative density of labelled protein band was analysed by Image‐ProPlus 5.0 software (Media Cybernetics Inc, Rockville, MD, USA)

Ou Q., Wang X., Wang Y., Wang Y, & Lin X. (2017). Oestrogen retains human periodontal ligament stem cells stemness in long‐term culture. Cell Proliferation, 51(2), e12396.

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Icariin Promotes Osteogenic Differentiation

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Icariin was purchased from Cayman (Ann Arbor, MI, USA). Fetal bovine serum (FBS) was purchased from Gibco Life Technologies (Grand Island, NY, USA). Dulbecco's modified Eagle's medium (DMEM), penicillin, and streptomycin were purchased from Hyclone (Hyclone, Waltham, MA, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Adipogenic and osteogenic differentiation media of rat bone marrow mesenchymal stem cells were purchased from Cyagen Biosciences Inc. (Santa Clara, CA 95050, USA). The total RNA extraction kit was purchased from OMEGA. The ReverTra Ace kit and SYBR Green Master Mix were purchased from TOYOBO (Tokyo, Japan). DKK-1, inhibitor of canonical Wnt signaling pathway, was purchased from R&D Systems (Minneapolis, MN, USA). The following antibodies used in the experiment were purchased from Cell Signaling Technology (Beverly, MA, USA): phospho-GSK-3β (#8213), β-catenin (#9562), Non-phospho (Active) β-Catenin (#8814), and GAPDH (#2118). Other reagents were of the highest commercial grade and were purchased from Sigma Chemical (St. Louis, MO, USA)

Huang J.M., Bao Y., Xiang W., Jing X.Z., Guo J.C., Yao X.D., Wang R, & Guo F.J. (2017). Icariin Regulates the Bidirectional Differentiation of Bone Marrow Mesenchymal Stem Cells through Canonical Wnt Signaling Pathway. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 8085325.

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Bone Morphogenetic Pathway Signaling Assay

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The Human Phospho-Kinase Array Kit and recombinant human BMP-2/ BMP-9 were purchased from R&D Systems (Minneapolis, MN, USA). The primary antibodies against anti-human Phospho-p53 (Ser6, 9, 15, 46, 392, and Thr81), Wnt5a/b, LRP6, Phospho-LRP6, Dvl2, Dvl3, Axin1, GSK3β, Phospho-GSK3β, PI3K, Phospho-PI3K, AKT, Phospho-AKT, MDM2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). The iScript cDNA Synthesis Kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). All primers and probes (RUNX2 #Hs00231692_m1; ALPL #Hs010291444_m1; BGLAP #Hs01587814_g1; NOG #Hs00271352_s1; GAPDH #Hs99999905-m1) were purchased from Applied Biosystems, Inc. (Bedford, MA, USA). Moreover, the TaqMan MicroRNA Reverse Transcription Kit and TaqMan Universal Master mix were from Applied Biosystems, Inc.

Park J.H., Koh E.B., Seo Y.J., Oh H.S, & Byun J.H. (2023). BMP-9 Improves the Osteogenic Differentiation Ability over BMP-2 through p53 Signaling In Vitro in Human Periosteum-Derived Cells. International Journal of Molecular Sciences, 24(20), 15252.

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Western Blot Analysis of Signaling Pathways

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Harvested cells were lysed in TBSN buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1.5 mM EDTA, 5 mM EGTA, 0.5% Nonidet P-40, and 0.5 mM Na₃VO₄) supplemented with proteinase inhibitors. The lysates were resolved by SDS-PAGE and transferred to Whatman Westran PVDF membrane (Sigma, Z671088), followed by incubation with antibodies against Plk1 (Santa Cruz, sc-17783), phospho-AKT-S473 (Cell Signaling, 9271), phospho-AKT-T308 (Cell Signaling, 13038), AKT (Cell Signaling, 9272), phospho-S6K (Cell Signaling, 9205), phospho-S6 (Cell Signaling, 2211), S6 (Cell Signaling, 2217), phospho-NFκB (Cell Signaling, 3033), NFκB (Santa Cruz, sc-372), AR (Santa Cruz, sc-7305), γ-tubulin (Sigma, T3559), p84 (Abcam, ab487), Twist1 (Sigma, SAB1411370), SREBP1 (Santa Cruz, sc-367), SREBP2 (Santa Cruz, sc-5603), phospho-GSK3β (Cell Signaling, 9322), GSK3β (BD Biosciences, 610201), FAS (BD Biosciences, 610962), HMG-CoA Reductase (EMD Millipore, ABS229), cleaved-PARP (EMD Millipore, AB3620), PSA (Cell Signaling, 5365), and β-actin (Sigma, A5441).

Zhang Z., Hou X., Shao C., Li J., Cheng J.X., Kuang S., Ahmad N., Ratliff T, & Liu X. (2014). Plk1 inhibition enhances the efficacy of androgen signaling blockade in castration-resistant prostate cancer. Cancer research, 74(22), 6635-6647.

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Protein Extraction and Western Blot Analysis

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Protein samples were purified from harvested cells in lysis buffer (100 mM Tris-HCl (pH 7. 5), 10 mM EDTA, 10 mM EGTA, 1% SDS, 20 mM NaCl (Sigma, St. Louis, MO)) containing 1 mM PMSF, Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific Inc., Waltham, MA). Lysates were centrifuged at 14,000 rpm for 30 min at 4 °C and supernatants collected and stored at −80 °C before use. Equal amounts of protein sample were used for Western blots as previously described [50 (link),28 (link)]. Western blots were performed with the following primary antibodies: PI3-kinase p85β (Santa Cruz Biotechnology, Santa Cruz, CA; 1:1,250); IRS-1, AKT, phospho-AKT, ERK, phospho-ERK, GSK-3β and phosphoGSK-3β (Cell Signaling; 1:1,250), 6E10 (Covance, Princeton, NJ, 1:1,000), 22C11 (Millipore, 1:1,000), and β-actin (Covance, 1:10,000). Alkaline phosphatase (AP)-conjugated anti-mouse or -rabbit secondary antibodies (Invitrogen, 1:2,500) and Immun-Star^™ AP Substrate (Bio-Rad, Hercules, CA) were used for protein detection. sAPPα in the media from PGK.sAPPα transduced PC12 cells was measured in Western blots using the 22C11 or 6E10 antibody. Quantification of immunoreactive bands was performed using Image J (NIH, http://rsb.info.nih.gov/ij/). Experiments were performed at least in triplicate for the same samples.

Kim W., Noh H., Lee Y., Jeon J., Shanmugavadivu A., McPhie D.L., Kim K.S., Cohen B.M., Seo H, & Sonntag K.C. (2014). MiR-126 Regulates Growth Factor Activities and Vulnerability to Toxic Insult in Neurons. Molecular neurobiology, 53(1), 95-108.

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Antibody Validation and Reagent Use

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Antibodies against the following proteins were used in this study: p62 (P0067, Sigma-Aldrich), LC3b (L7543, Sigma), GAPDH (60004-1-Ig, proteitech), FLAG (F3165, Sigma-Aldrich), GFP (AB513, Evrogen), p53 (ET1601–13, HuaAn Biotechnolog), phospho-Histone H3 (ab11477, abcam), phospho-Akt (#4060S, Cell Signaling), Akt (#9272, Cell Signaling), phospho-GSK-3β (#9323, Cell Signaling), GSK-3β (#9315, Cell Signaling), β-tubulin (ab6046, Abcam), HDAC1 (ab41407, Abcam) and zebrafish CD44a (25340-1hz, Abmart). Secondary antibodies for immunoblotting or immunofluorescence used in this study: goat anti-rabbit IgG and goat anti-mouse IgG (Prod #31460 and #31430, pierce), ReadyProbes™ Alexa Fluor® 594 Goat Anti-Mouse IgG Antibody (#R37121, Invitrogen), Alexa Fluor™ 488 Goat anti-Rabbit IgG (#A11008, Invitrogen) and Alexa Fluor™ 488 (#A110011, Invitrogen) Goat anti-Mouse IgG. The chemicals used in this study: Z-IETD-FMK (50 μm; #S7314, Selleck), pifithrin-μ (50 μm; #S2930, Selleck), pifithrin-α (5 μm; #S2929, Selleck), rapamycin (200 nm; #S1039, Selleck) and CQ (100 μm; #C6628, Sigma-Aldrich) and MitoTracker Deep red (Invitrogen™, M22426).

Cao L., Fang H., Yan D., Wu X.M., Zhang J, & Chang M.X. (2022). CD44a functions as a regulator of p53 signaling, apoptosis and autophagy in the antibacterial immune response. Communications Biology, 5, 889.

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Proteomic Analysis of Muscle Tissue

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Powdered muscle tissue was homogenized in RIPA buffer (Millipore, 20–188) supplemented with phosphatase inhibitor and protease inhibitor cocktail (Sigma-Aldrich). Lysates were subjected to SDS-PAGE and blotted using following antibodies: p110α (CS4249, 1:1000), phospho-Akt (S473) (CS9271, 1:2000), phospho-ERK (CS9101, 1:2000), phospho-GSK3β (CS9336, 1:2000), phospho-4EBP1 (CS2855, 1:1000), phospho-ULK (S555) (CS5869, 1:1000), phospho-Drp1 (S637) (CS4867, 1:1000), phospho-FoxO1/3 (CS9464, 1:500), ERK (CS9102, 1:2000), GSK3β (CS9315, 1:2000), 4EBP1(CS9644, 1:500), LC3 (CS2775, 1:1000), Beclin-1(CS3495, 1:1000), Drp1 (CS8570, 1:1000) and FoxO1 (CS2880, 1:500) from Cell Signaling, OPA1 (SC393296, 1:1000), GAPDH from Santa Cruz (SC25778, 1:5000), and citrate synthase (Ab96600, 1:1000), Mfn2 (Ab56889, 1:1000), and OXOPHOS (Ab110413, 1:1000) proteins from Abcam. Uncropped blots are available in Supplementary Fig. 8.

Li M.E., Lauritzen H.P., O’Neill B.T., Wang C.H., Cai W., Brandao B.B., Sakaguchi M., Tao R., Hirshman M.F., Softic S, & Kahn C.R. (2019). Role of p110a subunit of PI3-kinase in skeletal muscle mitochondrial homeostasis and metabolism. Nature Communications, 10, 3412.

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