Anti p gsk 3β

Manufactured by Abcam

Sourced in United Kingdom, United States

Anti-p-GSK-3β is an antibody that recognizes the phosphorylated form of glycogen synthase kinase 3 beta (GSK-3β). GSK-3β is a serine/threonine protein kinase that plays a key role in regulating various cellular processes. The phosphorylation of GSK-3β can modulate its enzymatic activity and downstream signaling pathways.

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Lab products found in correlation

Anti β catenin, by Abcam (8 mentions) Anti gsk3β, by Abcam (7 mentions) Anti bax, by Abcam (4 mentions) Anti bcl 2, by Abcam (4 mentions) Pvdf membrane, by Merck Group (3 mentions) Anti pi3k, by Abcam (2 mentions) Anti β actin, by Merck Group (2 mentions) Anti akt, by Cell Signaling Technology (2 mentions) Anti ki67, by Abcam (2 mentions) Anti gapdh, by Abcam (2 mentions) Anti akt, by Abcam (2 mentions) Anti p akt, by Abcam (2 mentions) Anti pcna, by Abcam (2 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Quantity one software, by Bio-Rad (1 mentions) Bca kit, by Beyotime (1 mentions) Anti rankl, by Abcam (1 mentions) Anti t pi3k, by Abcam (1 mentions) Anti opg, by Cell Signaling Technology (1 mentions) Anti p pi3k, by Abcam (1 mentions)

Close spelling variants of this product

GSK-3β (104 citations) P-GSK-3β (62 citations)

13 protocols using anti p gsk 3β

Proteomic Analysis of Bone Remodeling

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Total proteins were extracted by RIPA lysis buffer (Beyotime, Jiangsu, China). The protein concentration was quantified through a BCA Kit (Beyotime). Each 20 μg of protein was separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked using non-fat milk (5%) at room temperature for 1 h, which were subsequently incubated at 4° C overnight with the following primary antibodies: anti-OPG (1:100; Cell Signaling Technology, USA), anti-RANKL (1:1000; Abcam, USA), anti-RUNX2 (1:1000; CST), anti-BMP2 (1:1000; CST), anti-p-PI3K (1:1000; Abcam), anti-t-PI3K (1:1000; Abcam), anti-p-AKT3 (1:1000; Abcam), anti-t-AKT3 (1:1000; Abcam), anti- p-GSK3β (1:1000; Abcam) anti-β-catenin (1:5000; Abcam). Then, the secondary antibody was added and incubated with the membranes for 2 h. The signal of the protein bands was visualized using ECL Plus reagents and their densities were evaluated by the Quantity One software (Bio-Rad Laboratories). All the assays were performed in triplicate.

Zhao F., Xu Y., Ouyang Y., Wen Z., Zheng G., Wan T, & Sun G. (2021). Silencing of miR-483-5p alleviates postmenopausal osteoporosis by targeting SATB2 and PI3K/AKT pathway. Aging (Albany NY), 13(5), 6945-6956.

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Immunoblotting and qPCR Analysis of LSG

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At the end of the experiment, fresh LSG tissues were excised rapidly, washed with 0.9% saline, dissected into small portions, and maintained at -80°C until use. Western blot analysis was performed to examine protein expression levels of P-PI3K, PI3K, AKT2, P-GSK3β, and GSK3β in LSG tissue. The primary antibodies used were anti-P-PI3K (Bioss, Woburn, Massachusetts, USA), anti-PI3K (Abcam Trading (Shanghai) Company, Shanghai, China), anti-AKT2 (Biorbyt, Cambridge, Cambridgeshire, United Kingdom), anti-P-GSK3β (Abcam Trading (Shanghai) Company, Shanghai, China), and anti-GSK3β (Bioss, Woburn, Massachusetts, USA). Protein expression levels were normalized to β-actin (CST, Danvers, MA, USA). Real-time PCR was used to quantitatively describe the mRNA expression of IL-1β, IL-6, and TNF-α. For quantification, the expression levels of mRNAs were normalized to the reference gene GAPDH.

Wang Z., Li S., Lai H., Zhou L., Meng G., Wang M., Lai Y., Wang Z., Chen H., Zhou X, & Jiang H. (2019). Interaction between Endothelin-1 and Left Stellate Ganglion Activation: A Potential Mechanism of Malignant Ventricular Arrhythmia during Myocardial Ischemia. Oxidative Medicine and Cellular Longevity, 2019, 6508328.

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Western Blot Analysis of Apoptosis and Wnt Signaling Pathways

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A commercial RIPA lysis reagent (Catalog No: CW2333; Cwbio) was used to extract total proteins. After quantification using the BCA Protein Assay Kit (Catalog No: CW0014; Cwbio), 20 μg of the protein sample was added to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the protein bands. The separated proteins were transferred to a polyvinylidene fluoride membrane and blocked for 2 h in 5% milk at room temperature. Subsequently, the membrane was incubated overnight at 4°C with the primary antibody and the secondary antibody at room temperature for 2 h. Finally, the protein signals were scanned using an ECL kit (Abcam, USA). All antibodies were obtained from Abcam, including anti-Bax (Catalog No: ab32503; 1/2,000), anti-Bcl-2 (Catalog No: ab32124; 1/2,000), anti-Wnt (Catalog No: ab63934; 1/1,000), anti-Gsk-3β (Catalog No: ab131356; 1/1,000), anti-phosphorylated Gsk-3β (anti-p-Gsk-3β; Catalog No: ab75814; 1/10,000), anti-β-catenin (Catalog No: ab68183; 1/1,000), anti-c-Myc (Catalog No: ab168727; 1/1,000), anti-GAPDH (Catalog No: ab9485; 1/2,500), and HRP-labeled goat anti-rabbit IgG (Catalog No: ab205718; 1/5,000).

Du J., Zhuo Y., Sun X., Nie M., Yang J., Luo X, & Gu H. (2023). hsa_circ_0000285 sponging miR-582-3p promotes neuroblastoma progression by regulating the Wnt/β-catenin signaling pathway. Open Medicine, 18(1), 20230726.

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Western Blot Analysis of Striatal Proteins

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Striatal brain tissues from the MCA were lysed with radioimmunoprecipitation assay buffer (RIPA) containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Proteins were separated by SDS-PAGE and then transferred onto a nitrocellulose membrane. The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-p-GSK-3β (Tyr216, 1:1000, Abcam Inc., Cambridge, MA); anti-GSK-3β (1:1000, Abcam); anti-β-actin (1:5000, Sigma-Aldrich); anti-p-β-catenin (Ser33/37/Thr41, 1:2000, Cell Signaling Technology Inc., Danvers, MA); anti-β-catenin (1:1000, Abcam), anti-claudin-3 (1:2000, Santa Cruz, CA); anti-claudin-5 (1:2000, Santa Cruz); anti-p-Akt (Ser473, 1:2000, Cell Signaling); anti-Akt (1:2000, Cell Signaling); anti-ICAM-1 (1:1000, Abcam); anti-VCAM-1 (1:1000, Abcam); anti-IKK-β (1:2000, Santa Cruz); anti-NF-kB (1:2000, Santa Cruz); anti-HHE (1:1,000, Abcam); anti-Iba1 (1:1,000, Abcam); and anti-myeloperoxidase (MPO) (1:2000, Santa Cruz). Secondary antibodies conjugated with horseradish peroxidase were used, and immunoreactivity was visualized by chemiluminescence (SuperSignal Ultra, Pierce, Rockford, IL, USA). Bands of interest were analyzed and quantified using Scion Image.

Chen F., Wang W., Ding H., Yang Q., Dong Q, & Cui M. (2016). The glucagon-like peptide-1 receptor agonist exendin-4 ameliorates warfarin-associated hemorrhagic transformation after cerebral ischemia. Journal of Neuroinflammation, 13(1), 204.

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Protein Extraction and Western Blot Analysis

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Total proteins from the cells or tissues were extracted using a protein lysis buffer with PhosStop phosphatase inhibitor cocktail and protease inhibitor cocktail (both from Roche Diagnostics, Indianapolis, IN, USA). Lysates were denatured prior to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Hertfordshire, UK). The membranes were blocked with 5% nonfat milk at 20–25 °C for 1 h and then incubated with primary antibodies overnight at 4 °C. The following day, the membranes were washed and further incubated with secondary antibodies for 1 h. The immunoreactive signals were visualized using enhanced chemiluminescence reagents (ECL, Pierce, Rockford, USA). The following antibodies were used: anti-ARFGEF1 (Abcam, ab183747, Cambridge, MA, USA), anti-Ki-67 (Abcam, ab15580), anti-Vimentin (Cell Signaling, #3932, Beverly, MA, USA), anti-E-cadherin (Cell Signaling, #3195), anti-N-Cadherin (Cell Signaling, #13116), anti-Snail (Cell Signaling, #3879), anti-Slug (Cell Signaling, #9585), anti-ZEB1 (Cell Signaling, #3396), anti-Twist1 (Cell Signaling, #46702), anti-ZEB2 (Abcam, ab138222), anti-AKT (Cell Signaling, #4685), anti-p-AKT (Cell Signaling, #4060), anti-GSK-3β (Abcam, ab32391), anti-p-GSK-3β (Abcam, ab75745), and anti-β-actin (Sigma, A5441).

Han J., Zhang M., Nie C., Jia J., Wang F., Yu J., Bi W., Liu B., Sheng R., He G., Kong L., Zheng L., Pang R., Ding Z., Chen L., Guan Q., Pan S., Meng X., Xu J., Liu L, & Zhang J. (2019). miR-215 suppresses papillary thyroid cancer proliferation, migration, and invasion through the AKT/GSK-3β/Snail signaling by targeting ARFGEF1. Cell Death & Disease, 10(3), 195.

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Analyzing miRNA Expression and Protein Profiles in B16F10 Cells

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Total RNA was extracted using TRIzol total RNA isolation reagent, according to the manufacturer’s instructions. miRNAs were detected with stem-loop primers purchased from RiboBio (Guangzhou, China). GAPDH and U6 small nucleolar RNA was used for normalization. qPCR was performed using the FastStart Universal SYBR Green Master (ROX) on a Real-Time PCR System (Applied Biosystems, USA). Sequences of Real-Time PCR primers are listed in Additional file 1: Table S1.
After treatment with hAESC-CM or hAMSC-CM for 48 h, B16F10 cells were lysed in RIPA buffer, containing protease inhibitor cocktail and total proteins were subjected to 10% or 12% SDS-PAGE, then transferred to PVDF membranes, which were incubated with primary antibodies anti-β-actin, anti-LC3B, anti-P62 (CST, USA), anti-TRP1, anti-MITF (Sigma, USA), anti-PCNA (OriGene, USA), anti-AKT, anti-P-AKT, anti-β-Catenin, anti-GSK3β, anti-p-GSK-3β (Abcam, USA) at 4 °C overnight. The membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies. Images were quantified using a digital gel image analysis system TANON 5500 (Shanghai, China) and the band intensities were quantified by Tanon GIS software.

Wang X.Y., Guan X.H., Yu Z.P., Wu J., Huang Q.M., Deng K.Y, & Xin H.B. (2021). Human amniotic stem cells-derived exosmal miR-181a-5p and miR-199a inhibit melanogenesis and promote melanosome degradation in skin hyperpigmentation, respectively. Stem Cell Research & Therapy, 12, 501.

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Protein Extraction and Western Blot Analysis

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RIPA buffer was used to extract total protein from HUFs, human and rat urethral tissues. BCA protein assay (Thermo Fisher Scientific) was performed to detect the protein concentration. Equal amounts of protein (20 µg) were separated by 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore, USA). Next, the membranes were blocked in 5% fat-free milk for 2 h, and incubated with the following antibodies at 4 °C overnight: anti-Wnt3a (1:1000, Abcam), anti-β-catenin (1:1000, Abcam), anti-DKK1(1:1000, Abcam), anti-collagen I (1:1000, Abcam), anti-collagen III (1:1000, Abcam), anti-α-SMA (1:1000, Abcam), anti-p-GSK-3β (1:1000, Abcam), anti-GSK-3β(1:1000, Abcam), anti-p-Smad2(1:1000, Abcam), anti-p-Smad3 (1:1000, Abcam), anti-Smad2/3 (1:1000, Abcam) and anti-GAPDH (1:5000, Abcam, Cambridge, UK). Membranes were cut horizontally based on the differences of molecular weight. Then membranes washed with TBST (Beyotime Biotechnology, Shanghai, China) and incubated with corresponding secondary antibodies (goat anti-rabbit IgG H&L (HRP), 1:10000; rabbit anti mouse IgG H&L (HRP), 1:10000; Abcam, USA) at room temperature for 1 h. Additionally, membranes were stripped with Western Blot Stripping Buffer (Cell Signaling Technology), and re-probed with target proteins. Target blots signals were observed by an enhanced chemiluminescence-detecting kit (Thermo Fisher, MA, USA).

Huang S., Fu D., Wan Z., Huang Z., Li M., Li H, & Chong T. (2023). DKK1 Ameliorates Myofibroblast Differentiation in Urethral Fibrosis in Vivo and in Vitro by Regulating the Canonical Wnt Pathway. International Journal of Medical Sciences, 20(12), 1631-1643.

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Western Blot Analysis of Protein Expression

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Total protein was extracted from HaCAT cells, DFL cells, and skin tissues. Western blot was performed to detect the target proteins. Sixty micrograms of total protein was run on 10% denaturing SDS-PAGE gels, then transferred to nitrocellulose membranes (BioRad), which were incubated with primary antibodies anti-GAPDH (1:1000, rabbit monoclonal, Santa Cruz), anti-β-actin (1:1000, mouse polyclonal, CST), anti-Bcl-2 (1:1000, mouse monoclonal, Abcam), anti-Bax (1:1000, mouse monoclonal, Abcam), anti-PCNA (1:1000, mouse monoclonal, Abcam), anti-CK19 (1:1000, mouse monoclonal, Abcam), PI3K (1:1000, rabbit polyclonal, CST), P-PI3K (1:1000, rabbit polyclonal, CST), anti-AKT (1:1000, mouse monoclonal, Abcam), anti-P-AKT (1:1000, mouse monoclonal, Abcam), mTOR (1:1000, rabbit polyclonal, CST), P-mTOR (1:1000, rabbit polyclonal, CST), anti-β-Catenin (1:1000, mouse monoclonal, Abcam), anti-GSK3β (1:1000, mouse monoclonal, Abcam), and anti-P-GSK3β (1:1000, mouse monoclonal, Abcam) at 4 °C overnight. Blots were detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibody (Invitrogen) for 1 h at room temperature. Images were quantified using the Super Signal West Pico or Femto chemiluminescent detection system (Pierce).

Li J.Y., Ren K.K., Zhang W.J., Xiao L., Wu H.Y., Liu Q.Y., Ding T., Zhang X.C., Nie W.J., Ke Y., Deng K.Y., Liu Q.W, & Xin H.B. (2019). Human amniotic mesenchymal stem cells and their paracrine factors promote wound healing by inhibiting heat stress-induced skin cell apoptosis and enhancing their proliferation through activating PI3K/AKT signaling pathway. Stem Cell Research & Therapy, 10, 247.

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Histological Analysis of Neonatal Hypoxia-Ischemia

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Anesthetized pups were transcardially perfused with 0.1 M PBS followed by 4% formaldehyde solution (PFA) 48 hours after HI. The brains were then removed, postfixed (4% PFA, 4°C, 24 hr), and then transferred into a 30% sucrose solution for 2 days. The cryoprotected brains were sectioned at a thickness of 10 µm with a cryostat (LM3050S, Leica Microsystems Inc, IL, USA) for double fluorescence staining. The sections were then washed three times with 0.1 M PBS and were incubated with blocking solution (10% normal goat serum in 0.1 M PBS) for 2 hours at room temperature following 0.1%Triton X-100 (37°C, 30 min). Primary antibodies anti-GFAP (1:200, Millipore, Billerica, MA, USA), anti-vWF (1:200, Millipore, Billerica, MA, USA), anti-G-CSFR (1:100, Santa Cruz Biotechnology, Inc., CA, USA), anti-p-GSK-3β (Tyr216,1:200, abcam, Cambridge, MA,USA), anti-β-Catenin (1:200, abcam, Cambridge, MA, USA), anti-MPO (1:100, Santa Cruz Biotechnology, Inc., CA, USA), and DAPI (Vector Laboratories Inc. Burlingame, CA, USA) were applied (4°C, overnight).
The sections were then washed with 0.1 M PBS and incubated for 2 hours with secondary antibodies (1:200, anti-mouse IgG labeled with Alexa Fluor-488, anti-rabbit IgG labeled with Alexa Fluor-568, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) at room temperature. The stained slices were observed with an OLYMPUS BX51 microscope.

Li L., McBride W., Doycheva D., Dixon B.J., Krafft P.R., Zhang J.H, & Tang J. (2015). G-CSF Attenuates Neuroinflammation and Stabilizes the Blood-Brain Barrier via the PI3K/Akt/GSK-3β Signaling Pathway Following Neonatal Hypoxia-Ischemia in Rats. Experimental neurology, 272, 135-144.

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Protein Expression Analysis Techniques

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Western blot analysis and immunofluorescence were preformed as previously described⁴; the primary antibodies are listed in Table S1, including anti‐GAPDH (1:10 000, rabbit monoclonal, Abcam), anti‐PCNA (1:1000, mouse monoclonal, Abcam), anti‐Ki67 (1:1000, mouse monoclonal, Abcam), anti‐cyclin E1 (1:1000, rabbit monoclonal, Abcam), anti‐cyclin D1 (1:10 000, rabbit monoclonal, Abcam), anti‐cyclin A2 (1:2000, rabbit monoclonal, Abcam), anti‐cyclin B1 (1:2000, rabbit monoclonal, Abcam), anti‐cleaved‐PARP (1:10 000, rabbit monoclonal, Abcam), anti‐cleaved‐caspase‐3 (1:1000, rabbit polyclonal, CST), anti‐IGFBP3 (1:1000, rabbit monoclonal, CST), anti‐DKK‐1(1:1000, mouse monoclonal, Santa Cruz), anti‐DKK‐3 (1:1000, rabbit monoclonal, Abcam), anti‐P‐Gsk3β (1:1000, rabbit polyclonal, Abcam), anti‐Gsk3β (1:1000, mouse monoclonal, Abcam), anti‐β‐catenin (1:5000, rabbit monoclonal, Abcam), anti‐AKT (1:1000, mouse monoclonal, Abcam), anti‐P‐AKT (1:1000, mouse monoclonal, Abcam), anti‐PI3K (1:1000, rabbit polyclonal, Abcam), P‐PI3K (1:1000, rabbit polyclonal, CST), anti‐IGF‐1R (1:1000, rabbit monoclonal, CST), anti‐P‐IGF‐1R (1:1000, rabbit monoclonal, CST), anti‐N‐cadherin (1:5000, rabbit monoclonal, Abcam), anti‐Bcl‐2 (1:1000, mouse monoclonal, Abcam) and anti‐Bax (1:1000, mouse monoclonal, Abcam).

Liu Q., Li J., Zhang X., Liu Y., Liu Q., Xiao L., Zhang W., Wu H., Deng K, & Xin H. (2020). Human amniotic mesenchymal stem cells inhibit hepatocellular carcinoma in tumour‐bearing mice. Journal of Cellular and Molecular Medicine, 24(18), 10525-10541.

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