The PE-conjugated monoclonal fluorescent antibody p-GSK3β (Ser9; Cell Signaling, Danvers, MA, USA) was used to identify p-GSK3β (Ser9) expression. Cells were plated in six-well plates, incubated, and harvested after treatment. The cells were then fixed with fixation agent for 15 min at room temperature and washed with PBS. Next, they were permeabilized with permeability agent for 5 min and incubated with an antibody against p-GSK3β (Ser9) at 4°C for 30 min. The samples were then analyzed by flow cytometry.
P gsk3β
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Canada
P-GSK3β is an antibody that specifically recognizes the phosphorylated form of glycogen synthase kinase-3 beta (GSK3β) protein. GSK3β is a serine/threonine protein kinase that plays a key role in the regulation of various cellular processes, including metabolism, cell cycle, and cell survival. Phosphorylation of GSK3β at specific serine residues (such as Ser9) is an important regulatory mechanism that modulates its activity.
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Lab products found in correlation
Gsk3β, by Cell Signaling Technology (175 mentions)
P akt, by Cell Signaling Technology (131 mentions)
β catenin, by Cell Signaling Technology (60 mentions)
β actin, by Cell Signaling Technology (50 mentions)
Gapdh, by Cell Signaling Technology (39 mentions)
P erk, by Cell Signaling Technology (34 mentions)
Cleaved caspase 3, by Cell Signaling Technology (28 mentions)
Cyclin d1, by Cell Signaling Technology (24 mentions)
Pvdf membrane, by Merck Group (24 mentions)
Bcl 2, by Cell Signaling Technology (23 mentions)
P mtor, by Cell Signaling Technology (21 mentions)
β actin, by Santa Cruz Biotechnology (21 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (20 mentions)
β actin, by Merck Group (19 mentions)
Gapdh, by Santa Cruz Biotechnology (19 mentions)
E cadherin, by Cell Signaling Technology (19 mentions)
P erk1 2, by Cell Signaling Technology (19 mentions)
Vimentin, by Cell Signaling Technology (18 mentions)
N cadherin, by Cell Signaling Technology (16 mentions)
P β catenin, by Cell Signaling Technology (16 mentions)
310 protocols using p gsk3β
1
Quantifying Phosphorylated GSK3β in Cells
2
Immunofluorescence of β-catenin and pGSK3β
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Immunofluorescence detection was performed for β-catenin and pGSK3β using primary anti-β-catenin (1:100, Cell Signaling Technology, United States), pGSK3β (1:100, Cell signaling Technology, United States) and secondary antibodies (1:2000, ZSGB-BIO, China) according to previously described methods (Peng et al., 2019 (link)). Nuclei were stained with DAPI. Fluorescence images were taken with a fluorescence microscope (Olympus BX51, Japan).
3
Protein Extraction and Western Blot Analysis
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The total protein of proximal ileocecal tissue was extracted, and we measured the concentration by the spectrophotometer method, trim as 2 g/l, electrophoresis by 10% SDS-PAGE. After the film was transferred by the wet rotating method, it was put into the closed liquid, and the 2 h was closed on the shaking table. The first antibody was added to culture at 4°C overnight. After that, the second antibody was added to culture at room temperature overnight. After exposure in a darkroom, developing and fixing. The GAPDH was considered as a reference in this study. The relative antibodies (PTEN, PI3K, p-PI3k, AKT, p-AKT, GSK-3β and GAPDH) were purchased from Abcam (USA) and p-GSK-3β was purchased from Cell Signaling (USA). PTEN (ab32199, Abcam, USA); PI3K (ab32089, Abcam, USA); p-PI3k (ab182651, Abcam, USA); AKT(ab79463, Abcam, USA); p-AKT (ab38449, Abcam, USA); GSK-3β (ab93926, Abcam, USA), p-GSK-3β (mAb #5558, Cell Signaling, USA)
4
Immunoblotting for Insulin Signaling Pathway
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Specific antibodies for pAkt(Ser473)(#4060, 1:5000), pAkt(Thr308)(#4056, 1:2500), Akt(#4691, 1:5000), pINSR(#3024, 1:5000), INSRβ(#3025, 1:5000), p-p70-S6K(#97596, 1:2000), S6K(#9202, 1:5000), pFoxO1(S256)(#9461, 1:2000), FoxO1(#2880, 1:5000), pGSK3β(#5558, 1:5000), GSK3α/β(#5676, 1:5000), p-ERK1/2(T202/Y204)(#9101, 1:5000), ERK1/2(#4695, 1:5000), and Hsp70(#4872, 1:2000) were obtained from (Cell Signaling Technology Inc., Danvers, MA, USA). pIRS1(#09432, 1:2500) and IRS1(#06248, 1:2500) were acquired from (Merck KGaA, Darmstadt, Germany). β-Actin(#A228, 1:5000) was procured from (Sigma-Aldrich). Peroxidase-conjugated secondary antibodies Anti-Rabbit IgG (#NA934, 1:5000) and Anti-Mouse IgG (#NA931, 1:5000) were purchased from (GE Healthcare BioSciences AB, Chicago, IL, USA). Anti-Rabbit IgG Secondary Antibody (Alexa Fluor™ 568, 1:5000) was obtained from (Thermo Fisher Scientific, Waltham, MA, USA).
5
Western Blot Analysis of Cell Signaling Proteins
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Proteins from total cell lysates were separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and probed with different primary antibodies against phospho (p)‐AKT (Ser473; Cell Signaling Technology), AKT (Cell Signaling), p‐GSK‐3β (Ser256; Cell Signaling Technology), GSK‐3β (Cell Signaling Technology), Nrf2 (Abcam), HO‐1 (heme oxygenase‐1; Cell Signaling Technology), NQO‐1 (NAD(P)H quinone dehydrogenase 1; Cell Signaling) and GAPDH (glyceraldehyde‐3‐phosphate dehydrogenase; Abcam). Of note, we only showed cleaved‐caspase 3 bands in figures instead of total caspase 3 and cleaved‐caspase 3 bands in the same membrane; the reason for this is that when we exposure total‐caspase 3 and cleaved‐caspase 3 at the same time, the cleaved‐caspase 3 band was undetectable, which has been described in our previous study.12
6
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Samples were lysed with RIPA containing 1% protease and phosphate inhibitor cocktail (P8340; Sigma-Aldrich). Proteins were run on 10% SDS-PAGE and then transferred to PVDF membranes, which were incubated with primary antibodies at 4°C overnight and probed by secondary antibodies. Blots were visualized with enhanced chemiluminescence (WBKLS0500; Millipore, Temecula, CA) and the ChemiDoc Touch detection system (Bio-Rad). The following primary antibodies were used: slug (9585; Cell Signaling Technology, Danvers, MA; 1:1000), β-catenin (8480; Cell Signaling Technology; 1:1000), p-eIF2α (3398; Cell Signaling Technology; 1:1000), p-GSK3β (5558; Cell Signaling Technology; 1:1000), GAPDH (ab8245; Abcam, Cambridge, England; 1:1000), C/EBPβ (ab32358; Abcam; 1:1000), N-cadherin (ab76057; Abcam; 1:1000), MMP-2 (ab92536; Abcam; 1:1000), AKT (ab8805; Abcam; 1:1000), p-AKT (ab81283; Abcam; 1:1000), vimentin (ab92547; Abcam; 1:5000), and cleaved caspase-3 (19677-1-AP; Proteintech, Rosemont, IL; 1:500).
7
Comprehensive Protein Expression Analysis
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For western blotting, cells were lysed buffer including 1% Triton X-100 and 1% NP-40, as well as the following protease and phosphatase inhibitors. The first step is to separate the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then, proteins can be electrophoretically transferred to PVDF membranes. The membranes were immunostained with primary antibodies. Furthermore, HRP-conjugated secondary antibodies are added. Detection was carried out using an enhanced chemiluminescence reagent (Amersham Biosciences, Buckinghamshire, UK). Antibodies against cleaved PARP, cleaved caspase-3, XIAP, Mcl-1, p-mTOR, p-AKT (Tyr308), p-4EBP1, p-GSK3β, and β-actin were purchased from Cell Signaling Technology (Danvers, MA), Abcam (Cambridge, MA), and Santa Cruz Biotechnology (Santa Cruz, CA).
8
Western Blot Analysis of Protein Targets
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Western analysis was performed as described previously (15 (link), 16 (link)). Target proteins were detected by using specific antibodies against BPTF (A300-973A at 1:1000 dilution) (Bethyl Laboratories, Montgomery, TX), pAKT (Ser473, #9271 at 1:500 dilution), total AKT (#4685, at 1:500 dilution), pGSK-3β (Ser9, #9323 at 1:500 dilution), CCND1 (#2978 at 1:500), BCL2 (#15071 at 1:500) (Cell Signaling Technology, Danvers, MA) and GAPDH (AB2302 at 1:10000 dilution, EMD Millipore, Billerica, MA).
9
Molecular Signaling Pathways Regulation
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Piperine (≥ 97% purity, as determined by high-performance liquid chromatography) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Isoprenaline (ISO, I5627), TGF-β (T7039), angiotensin II (Ang II, A9525) and phenylephrine (PE, P6126) were purchased from Sigma-Aldrich. The primary antibodies against the following proteins were purchased from Cell Signaling Technology: T-AKT (4691, 1:1000), phospho-AKT (P-AKT, 4060, 1:1000), T-glycogen synthase kinase 3β (GSK3β, 9315, 1:1000), P-GSK3β (9323P, 1:1000), T-P38 (9212P, 1:1000), P-P38 (4511P, 1:1000), T-AMPKα (2603P, 1:1000), P-AMPKα (2535, 1:1000), T-ERK (4695, 1:1000), P-ERK (4370P, 1:1000), T-SMAD3 (3103S, 1:500), P-SMAD3 (3101, 1:500) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 2118, 1:1000). α-Smooth muscle actin (α-SMA, ab7817, 1:500) was obtained from Abcam (Cambridge, UK). Antibodies against α-actinin (ab90776) were obtained from Merck Millipore (Massachusetts, United States), and anti-vimentin (sc-5565) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-rabbit/mouse EnVisionTM +/HRP reagent used for immunohistochemistry was purchased from Gene Technology (Shanghai, China), and Alexa Fluor 488-goat anti-mouse secondary antibody was purchased from LI-COR Biosciences (Lincoln, USA). The BCA protein assay kit was from Pierce (Rockford, IL, USA).
10
Western Blot Analysis of Cellular Signaling
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Cell lysates were extracted with CETi protein extraction solution (TransLab, Daejeon, Korea). Thirty to fifty micrograms of protein extract was used for western blotting with antibodies against p-Rbs, p21, cyclin D1, GSK3β, p-GSK3β, CatB, GS, p-GS, p38MAPK, p-p38MAPK, p70S6K, p-p70S6K (Cell Signaling, Denvers, MA, USA), α-tubulin, mouse IgG peroxidase conjugate, rabbit IgG peroxidase conjugate (Sigma-Aldrich Corp.) and Rb (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The human phospho-MAPK Array Kit (R&D Systems, Inc., Minneapolis, MN, USA) was used according to the manufacturer's instructions. A polyclonal antibody against CST1 was obtained from mice immunized with the purified protein.
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