P gsk3β

Manufactured by Santa Cruz Biotechnology

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P-GSK3β is a phosphorylated form of the enzyme Glycogen Synthase Kinase 3 beta (GSK3β). GSK3β is a serine/threonine protein kinase that plays a key role in the regulation of various cellular processes, including metabolism, cell proliferation, and apoptosis. The phosphorylation of GSK3β at specific serine residues can modulate its enzymatic activity and function.

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P‐GSK3β Ser9 (12 citations)

34 protocols using p gsk3β

Exosome Isolation and GSK3β Phosphorylation Analysis

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The isolation of exosomes was approved by western blot analysis using the antibody of CD63 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Also, the phosphorylation of GSK3β at Ser-9 position was determined in the cells treated with the doses of 2 and 4 µg/mL the pooled exosomes obtained from normal-weight (PN-Exo) females and PO-Exo as well as in the cells treated with the dose of 4 µg/mL obese exosome (O-Exo) and normal-weight exosome (N-Exo) using the western blot analysis. Briefly, the cells were lysed by RIPA buffer (50 mM Tris–HCl, 1% Triton X-100, pH 7.4, 0.2% SDS, 0.2% sodium deoxycholate, 1 mM PMSF and 1 mM Na-EDTA) containing protease inhibitor cocktail and then, the cell lysate was fractionated using SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Five percent non-fat dry milk was applied to blocking. Immunoblots were incubated with antibodies against GAPDH, p-GSK3β, and GSK3β (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 18 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 60 min. The bound proteins were observed by chemiluminescence using enhanced electrochemiluminescence (ECL) reagents and subsequent autoradiography. Eventually, the protein bands were quantitated using ImageJ software.

Afrisham R., Sadegh-Nejadi S., Meshkani R., Emamgholipour S, & Paknejad M. (2020). Effect of circulating exosomes derived from normal-weight and obese women on gluconeogenesis, glycogenesis, lipogenesis and secretion of FGF21 and fetuin A in HepG2 cells. Diabetology & Metabolic Syndrome, 12, 32.

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Immunofluorescent Analysis of Cartilage Markers

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The sectioned samples were washed 3 times with PBS, and permeabilized with 0.25% Triton X-100 (Sigma). Samples were then incubated in 1% BSA, 22.5 mg/ml glycine in PBST (1 × PBS and 0.1% Tween 20) at room temperature for 1 hour to block non-specific binding, followed by overnight incubation at 4 °C with primary antibodies (pGSK-3β, Santa Cruz Biotechnology; sc-373800, pP38α, Cell Signaling Technologies, 9211S, collagen type II, abeam, ab34712, aggrecan, abeam, ab3778, collagen type V1, sc-20649). The samples were then washed 3 times with PBS and incubated in the dark at room temperature for 1 hour with secondary antibodies (such as goat anti-mouse IgG Dylight 488, goat anti-rabbit IgG Dylight 594 in 1:200 dilution). All the antibodies were diluted in 1% BSA in PBST. Finally, samples were washed and stained for nuclei using DAPI and/or F-actin using phalloidin (1:40 dilution). Samples were imaged using Zeiss LSM 880 laser scanning confocal microscope. The obtained images were analyzed using FIJI-imageJ v1.52i software.

Agarwal P., Lee H.P., Smeriglio P., Grandi F., Goodman S., Chaudhuri O, & Bhutani N. (2021). A dysfunctional TRPV4-GSK3β pathway prevents osteoarthritic chondrocytes from sensing changes in extracellular-matrix viscoelasticity. Nature biomedical engineering, 5(12), 1472-1484.

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Western Blot Protein Analysis

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The detailed process of western blot analysis can be found in a previous study (19 (link)). The antibodies used were: Flt1, cyclin D1, p21, matrix metalloproteinase 2 (MMP2), MMP9, E-cadherin, vimentin, VE-cadherin, Wnt1, β-catenin, GSK-3β, p-GSK-3β and β-actin were all from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Wang Q., Xu B., Du J., Xu X., Shang C., Wang X, & Wang J. (2018). MicroRNA-139-5p/Flt1/Wnt/β-catenin regulatory crosstalk modulates the progression of glioma. International Journal of Molecular Medicine, 41(4), 2139-2149.

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MCP-1 Protein Expression and Signaling

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Human recombinant monocyte chemoattractant protein 1 (MCP-1) protein was purchased from PeproTech (Rocky Hill, NJ, USA). Anti-rabbit and anti-mouse IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies (specific for p-GSK3β (Santa Cruz sc-135653) and GSK3β (Santa Cruz sc-9166)), and mouse monoclonal antibodies (specific for VEGF-A (Santa Cruz sc-7269), β-actin (Santa sc-47778), MCP-1 (Santa Cruz sc-32771), ILK (Santa Cruz sc-20019), p-MEK1/2 (Santa Cruz sc-271914), MEK1/2 (Santa Cruz sc-6250), p-ERK1/2 (Santa Cruz sc-7383), and ERK1/2 (Santa Cruz sc-1647)) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The control miRNA and miR-29c mimic were purchased from Life Technologies (Carlsbad, CA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Lien M.Y., Chang A.C., Tsai H.C., Tsai M.H., Hua C.H., Cheng S.P., Wang S.W, & Tang C.H. (2020). Monocyte Chemoattractant Protein 1 Promotes VEGF-A Expression in OSCC by Activating ILK and MEK1/2 Signaling and Downregulating miR-29c. Frontiers in Oncology, 10, 592415.

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Myricitrin Isolated from Myriace rubrae

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Myricitrin was isolated from the grounded barks of Myriace rubrae and was identified by Professor Rui-le Pan. The purity is over 99%, as detected by high-performance liquid chromatography (UV and DAD)41 (link). Metformin (Sino-American Shanghai Squibb Pharmaceuticals Ltd,) was used as the positive control in this study. All cell culture materials, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Gibco (NY, USA). The kits for determining the CK, LDH and AST enzyme levels were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Akt inhibitor (LY294002) was purchased from Selleckchem (TX, USA). Primary antibodies against Akt, p-Akt, GSK-3β, p- GSK-3β, P65, p-IKKβ, TNF-α, ERK, p-ERK, TGF-β1, Collagen I, Caspase-3, Caspase-9, Bcl-2, Bax, Nrf2, HO-1, NQO-1, γ-GCS, Lamin B1 and β-actin were obtained from Santa Cruz Biotechnology (CA, USA). FITC-anti-rabbit IgG second antibody was purchased from Molecular Probes (Life Technologies, CA, USA).

Zhang B., Shen Q., Chen Y., Pan R., Kuang S., Liu G., Sun G, & Sun X. (2017). Myricitrin Alleviates Oxidative Stress-induced Inflammation and Apoptosis and Protects Mice against Diabetic Cardiomyopathy. Scientific Reports, 7, 44239.

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Western Blot Analysis of Apoptotic Proteins

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Heart tissues were frozen, suspended in lysis buffer and phenylmethanesulfonyl fluoride (PMSF) at 4℃ for 30 minutes, boiled at 97℃ for 10 min, and then centrifuged for 10 min (13,000 rpm, 4℃). A bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) was utilized to measure the protein concentration in the supernatant. The proteins were resolved and transferred to poly vinylidene fluoride (PVDF) membranes, and Western blotting was performed using antibodies against bcl-2, bax, p-Akt, Akt, p-GSK-3β, GSK-3β, and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h and developed with an ECL kit (Pierce Biosciences, USA). The bands were visualized using an enhanced chemiluminescence reagent kit (Pierce Biosciences, USA). Immunoreactive bands were visualized by enhanced chemiluminescence, and densitometry was performed using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Wang C.Y., Li X.D., Hao Z.H, & Xu D. (2016). Insulin-like growth factor-1 improves diabetic cardiomyopathy through antioxidative and anti-inflammatory processes along with modulation of Akt/GSK-3β signaling in rats. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 20(6), 613-619.

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Apoptosis Regulation via Substance P

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Substance P and NK-1 antagonist were purchased by Sigma (USA); antibodies for β-catenin, p-GSK-3β, c-myc, and cleaved caspase-3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cell growth medium was composed of L-DMEM (90%, Gibco, USA) and fetal bovine serum (FBS, 10% v/v, Gibco, USA). The Annexin V-FITC apoptosis detection kit was obtained from KeyGEN Biotech Co., Ltd. (Guangzhou, China).

Fu S., Jin D., Liu S., Wang L., Wang Z., Mei G., Zou Z.L., Wu J.Q, & Xu Z.Y. (2015). Protective Effect of Neuropeptide Substance P on Bone Marrow Mesenchymal Stem Cells against Apoptosis Induced by Serum Deprivation. Stem Cells International, 2015, 270328.

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Genistein and Isoflurane Neuroprotective Mechanisms

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Genistein and isoflurane (0.75%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). For expression analysis, antibodies against CREB, p-CREB, cleaved caspase-3, Bcl-2, Bad, Bcl-xL, Bax, β-actin, phosphatase and tensin homolog (PTEN), and mammalian target of rapamycin complex 1 (mTORc1) were purchased from Cell Signaling Technology (Beverly, MA, USA). Akt, p-Akt, GSK-3β, p- GSK-3β, BDNF, TrkB, and p-TrkB were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PDE4 and Ca²⁺/calmodulin-dependent kinase IV (CaMKIV) were from Abcam. All other chemicals and reagents used in the present study were purchased from Sigma-Aldrich unless otherwise noted.

Jiang T., Wang X.Q., Ding C, & Du X.L. (2017). Genistein attenuates isoflurane-induced neurotoxicity and improves impaired spatial learning and memory by regulating cAMP/CREB and BDNF-TrkB-PI3K/Akt signaling. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 21(6), 579-589.

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Osteogenic Differentiation: Protein Analysis

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After 7 days of osteogenic induction, cells were washed with PBS and total protein was obtained by RIPA lysis buffer (Beyotime, China). Quantitative analysis was performed using BCA (Sigma, USA). Next, proteins were isolated on NuPAGE 10–12% polyacrylamide gel and transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% milk for 2 h and then incubated with primary antibodies overnight. The following primary antibodies were used: ALP (ABCAm, 1:400), RUNX2 (ABCAm, 1:400), total-β-catenin (ABCAm, 1:1000), active-β-catenin (Cell Signaling, 1:500), GSK-3β (ABCAm, 1:1000), p-GSK-3β (Santa Cruz, 1:500) and GAPDH (Cwbiotech, 1:500). The secondary antibody (Corning, 1:5000) was incubated according to the source of primary antibody, and the chemiluminescence ECL kit (Sigma, USA) was used for protein detection. ImageJ was used to analyze the corresponding spectral band intensity of scanned images.

Li Y., Liu A., Zhang L., Wang Z., Hui N., Zhai Q., Zhang L., Jin Z, & Jin F. (2021). Epithelial Cell Rests of Malassez Provide a Favorable Microenvironment for Ameliorating the Impaired Osteogenic Potential of Human Periodontal Ligament Stem Cells. Frontiers in Physiology, 12, 735234.

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Analysis of Glomerular Protein Expression

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Cultured cells were lysed and isolated renal glomeruli were homogenized in RIPA buffer supplemented with protease inhibitors. Nuclear fractions of cultured cells or isolated glomeruli were prepared by using the NE-PER kit (Thermo Scientific, Rockford, Illinois, USA). Samples were subjected to immunoblot analysis as previously described.^{28 (link)} The antibodies against GSK3β, p-GSK3β, synaptopodin, cathepsin L, GAPDH were purchased from Santa Cruz Biotechnology and those against p-RelA/p65 serine 467, p-RelA/p65 serine 536, p-RelA/p65 serine 276, cleaved caspase 3, β-catenin, histone H3 were purchased from Cell Signaling (Beverly, MA, USA). The antibodies against B7-1 and MCP-1 was purchased from R&D Systems (Minneapolis, Minnesota, USA).

Bao H., Ge Y., Peng A, & Gong R. (2015). Fine-tuning of NFκB by Glycogen Synthase Kinase 3β directs the fate of glomerular podocytes upon injury. Kidney international, 87(6), 1176-1190.

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