Western blotting and immunofluorescent assays were performed according to corresponding standard methods [25 (link)]. The specific antibodies used were listed as follows: PTEN (Cell Signaling Technology (CST), 1:1000 dilution), GSK3β (CST, 1:800 dilution), Akt (CST, 1:800 dilution), phospho (p) Akt (Ser473) (CST, 1:500 dilution), pGSK3β (Ser9) (CST, 1:500 dilution), GAPDH (CST, 1:5000 dilution), β-catenin (Abcam, 1:50 dilution), and Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1:200, 2 mg/mL, Invitrogen). Fluorescence images were captured by a laser scanning confocal microscope (Zeiss, Germany).
P gsk 3β
Manufactured by Abcam
Sourced in United States, United Kingdom
P-GSK-3β is a recombinant protein that represents the phosphorylated form of Glycogen Synthase Kinase-3 beta (GSK-3β). GSK-3β is a serine/threonine protein kinase involved in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Gsk 3β, by Abcam (43 mentions)
β catenin, by Abcam (22 mentions)
P akt, by Abcam (19 mentions)
Gapdh, by Abcam (13 mentions)
Pvdf membrane, by Merck Group (10 mentions)
E cadherin, by Abcam (9 mentions)
N cadherin, by Abcam (9 mentions)
P mtor, by Abcam (8 mentions)
Vimentin, by Abcam (7 mentions)
Bcl 2, by Abcam (6 mentions)
Cyclin d1, by Abcam (6 mentions)
α sma, by Abcam (6 mentions)
β actin, by Abcam (6 mentions)
Bcl2 associated x (bax), by Abcam (5 mentions)
C myc, by Abcam (5 mentions)
P akt, by Cell Signaling Technology (5 mentions)
E cadherin, by Cell Signaling Technology (4 mentions)
β catenin, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
62 protocols using p gsk 3β
1
Protein Quantification and Localization
2
Western Blot Analysis of Phosphorylated Kinases
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Total protein was extracted from the cultured cells, and then subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred onto 0.45 μm PVDF membranes (Millipore). Membranes were then blocked in 5% milk-TBST for 1 h and incubated with primary antibodies p-AKT, p-GSK3α, p-GSK3β (diluted 1:1000), and AKT, GSK3α, GSK3β, β-actin (diluted 1:5000 in TBST) overnight. After appropriate secondary antibodies for 1 h at room temperature, the membranes were detected by Tanon 5200 Imaging System (Shanghai, China). Antibodies were purchased as follows: p-AKT (Abcam, Cat. ab81283), AKT (Abcam, Cat. ab179463), p-GSK3α (Abcam, Cat. ab131112), GSK3α (Abcam, Cat. ab40870), p-GSK3β (Abcam, Cat. ab75814), GSK3β (Abcam, Cat. ab32391).
3
Western Blot Analysis of Apoptosis and Cell Cycle Markers
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For western blot analysis, cells were harvested and extracted in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.1% SDS, 1% NP-40, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). The analysis was performed as described previously [17 ]. The primary antibodies used to detect apoptotic markers were Bcl-2 (Santa Cruz, California, CA, USA), Bax (Abcam, Cambridge, MA, USA), cytochrome C (Santa Cruz, California, CA, USA), PARP, cleaved PARP, caspase 3, cleaved caspase 3, caspase 7, cleaved caspase 7, caspase 9, and cleaved caspase 9 (all from CST, Danvers, MA, USA). The primary antibodies c-Myc, cyclin D1, and cyclin E1 (all from Abcam, Cambridge, MA, USA) were used to detect cell cycle proteins. β-Catenin, GSK3β, p-GSK3β, c-Myc, cyclin D1, and cyclin E1 (all from Abcam, Cambridge, MA, USA) were used to analyze the Wnt/β-catenin pathway. β-Actin (CST, Danvers, MA, USA) was used as an internal control.
4
Cell Cycle Regulation and Apoptosis Signaling Assay
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Wst-1 assay kit was purchased from Daeillab (Daeillab, Korea). LY294002 (PI3K/Akt inhibitor) and PD98059 (Erk inhibitor), L-Ascorbic acid were purchased from Sigma Aldrich (Sigma Aldrich, USA). BIO (GSK-3β inhibitor) was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Specific antibodies such as p-Akt, total-Akt, β-actin, Cyclin E, p-cdk2, p21 were obtained from Cell Signaling Technology (Beverly, USA). and p-GSK-3β, total-GSK-3β, p-Erk, total-Erk, Active-caspase-3 antibodies were purchased from Abcam (Cambridge, USA). Muse™ Muse™ Cell Cycle Kit (MCH100106) and Muse™ Cell Analyzer (PB4455ENEU) were purchased from Millipore (EMD Millipore Corporation, Germany). 3M™ Tegaderm (sterile barrier to external contaminants) was purchased from 3 M (3 M, USA).
5
Protein Expression Analysis Workflow
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The total protein concentrations in cells were determined using a BCA protein assay kit (Thermo Fisher Scientific). The cells were lysed in RIPA lysis buffer (Millipore Corporation, MA, USA), and equal amounts of protein were separated by SDS-PAGE gels and transferred to PVDF membranes (Merck Millipore, MA, USA). The blots were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. The membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with HRP-linked secondary antibodies at 37 °C for 1 h. Western blotting was performed to evaluate the specific protein of interest based on proteomic results and its related proteins in the pathway, including CAV-1 (1:1000; Abcam), β-catenin (1:1000; Abcam), p-GSK-3β (1:1000; Abcam), GSK-3β (1:1000; Abcam), and VEGF (1:1000; Abcam). The immunoreactive bands were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific) and quantified using ImageJ software.
6
Protein Expression Analysis of Signaling Pathways
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Cells that were treated as described above were lysed in RIPA buffer (Cell Signaling Technology, MA) to extract the total proteins in line with the manufacturer’s instructions. The proteins were isolated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin (BSA) and incubated respectively with NR3C1 antibody (Origene, MD; 1:800), AKT (Invitrogen, New York, NY; 1:1000), p-AKT (Thr308) (Invitrogen, New York, NY; 1:800), p-AKT (Ser473) (Invitrogen; 1:500), mTOR (Abcam, MA; 1:800), p-mTOR (Abcam; 1:1000), GSK-3β (Abcam; 1:800), p-GSK-3β (Abcam; 1:1000), and β-Actin (Santa Cruz, CA; 1:1000). The membrane was then incubated with a corresponding secondary antibody. Enhanced chemiluminescence (Pierce, Rockford, IL) was used to detect the antigen-antibody complex.
7
Arctigenin Modulates Epithelial-Mesenchymal Transition
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Arctigenin was obtained from Nanjing Zelang Medical Technology Co. Ltd. (Nanjing, China). Paraquat dichloride was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against the following proteins were used: Occludin, α-SMA, Wnt3a, β-Catenin, GSK-3β, P-GSK-3β, β-actin (Abcam, United States). Cytokeratin 18(Proteintech group Inc., United States), E-cadherin, Vimentin (Cell Signaling Technology, Beverly, MA, United States), Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit IgG were obtained from Santa Cruz Biotechnogy (Santa Cruz, CA, United States). HYP kit (Nanjing Jiancheng Bioengineering Institute) Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were provided by Gibco (Rockville, MD, United States). All other chemicals and reagents in this study were of analytical grade.
8
Protein Expression Analysis in Kidney
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The podocytes and kidney tissues were lysed, and equivalent amount of protein (20 g) per sample was separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skimmed milk in the TBST buffer, the membranes were incubated with antibodies against nephrin, α-smooth muscle actin (α-SMA), beclin-1, p62, AKT, P-Akt, GSK3β, p-GSK3β (all from Abcam, UK), and LC3-II (Sigma). The blots were washed and incubated with the HRP-conjugated secondary antibody and developed using chemiluminescence reagents.
9
Western Blot Analysis of Cell Signaling Proteins
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Cells were lysed with the RIPA extraction reagent (Beyotime, China) supplemented with protease inhibitors (Sigma-Aldrich, USA). Equal amounts of total protein were separated using 10-12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.45 μm PVDF membrane (Millipore, USA). The membranes were blocked with 5% nonfat dry milk in TBST for 2 hours at room temperature and then incubated with specific primary antibodies overnight at 4°C followed by incubation with HRP-conjugated secondary antibodies at 37°C for 2 h at room temperature. Immune response bands were exposed via a Bio-Rad ChemiDoc XRS+ Imaging System (Bio-Rad, USA). Primary antibodies were DTL (Abcam, cat# ab72264), P21(CST, cat# 2947), CDK1(Abcam, cat# ab133327), CDK2 (Abcam, cat# ab32147), CDK4 (CST, cat# 12709), cyclinB1 (CST, cat# 12231S), CDT1(Abcam, cat# ab202067), SETD8(Abcam, cat# ab230683), E-cadherin (CST, cat# 3195S,), N-cadherin (CST, cat# 13116S), AKT (CST, cat# 4691L), p-AKT (CST, cat# 4060L,), GSK-3β (CST, cat# 12456S), p-GSK-3β (CST, cat# 5558S), mTOR (Abcam, cat# ab32028), p-mTOR (Abcam, cat# ab109268), and GAPDH (Santa Cruz, cat# sc-365062) antibodies.
10
Western Blotting of Cardiac Proteins
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The Western blotting protocol was described in our previous study (Zhao et al., 2016 (link)). The total protein of the heart tissue was isolated from homogenized tissues with TRIzol reagent (Invitrogen, Carlsbad, CA) using standard Invitrogen protocols. After quantifying with bicinchoninic acid (BCA), the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk, the membrane was incubated overnight in primary antibody (FGF1, AKT, p-AKT, GSK-3α/β, p-GSK-3β, Nrf2, SOD2, NQO1, 1:1000, Abcam). The membranes were then washed with TBST and incubated with specific horseradish peroxidase-conjugated secondary antibody. Quantity One software was used to analyze the blots after detection using enhanced chemiluminescence system.
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