RPPA as performed in our laboratory has been described previously (14 (link), 15 (link)) and was used to quantify PTEN expression and phosphorylation of AKT at Thr308 and Ser473, glycogen synthase kinase 3 (GSK3) at Ser21, mammalian target of rapamycin (mTOR) at Ser2448, and p70S6K at Thr389 as a ratio to total expression of each protein using antibodies from Cell Signaling (AKT, PTEN, mTOR and all phospho-specific antibodies), Epitomics, Inc. (total p70S6K antibody), and Santa Cruz Biotechnology (total GSK3 antibody). Because of potential effects of differences in tissue handling on protein phosphorylation in particular, 306 tumors from a single-institution batch (Clinic Hospital) were used for this analysis.
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Chemicals & Drugs
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Amino Acid
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Glycogen Synthase Kinase 3
Glycogen Synthase Kinase 3
Glycogen Synthase Kinase 3 is a serine/threonine protein kinase that plays a key role in the regulation of glycogen metabolism, cell signaling, and development.
It is invovled in the phosphorylation and inactivation of glycogen synthase, a rate-limiting enzyme in glycogen synthesis.
Glycogen Synthase Kinase 3 has also been implicated in the pathogenesis of various diseases, including diabetes, neurodegenerative disorders, and cancer.
Understanding the function and regulation of this enzyme is crucial for the development of targeted therapies.
It is invovled in the phosphorylation and inactivation of glycogen synthase, a rate-limiting enzyme in glycogen synthesis.
Glycogen Synthase Kinase 3 has also been implicated in the pathogenesis of various diseases, including diabetes, neurodegenerative disorders, and cancer.
Understanding the function and regulation of this enzyme is crucial for the development of targeted therapies.
Most cited protocols related to «Glycogen Synthase Kinase 3»
Antibodies
Antibodies, Phospho-Specific
Glycogen Synthase Kinase 3
Immunoglobulins
mTOR Protein
Neoplasms
Phosphorylation
Proteins
PTEN protein, human
Ribosomal Protein S6 Kinases, 70-kDa
Staphylococcal Protein A
Tissues
Cells were cultured without feeders or serum, unless specifically noted, in N2B27 medium prepared as described2 (link) or preformulated (NDiff™ N2B27 base medium, Stem Cell Sciences Ltd, Cat. No. SCS-SF-NB-02) supplemented with Small molecule inhibitors PD0325901 (PD, 1 μM) and CHIRON99021 (CH, 3 μM) and LIF prepared in house (2i+LIF). Cells were routinely propagated on 0.1% gelatine-coated plastic and replated every 3 days at a split ratio of 1 in 10 following dissociation with Accutase (Gibco). Alternative Gsk3 inhibitors were provided by Pfizer: Compounds A (750 nM), B (50 nM), C (1 μM), D (250 nM), E (100 nM), F (150 nM) and G (50 nM) are described, see Supplementary Information , Figure 1 . Colony forming assays were performed by plating 600 ES cells per well on laminin (Sigma) coated plates. Plates were fixed and stained for alkaline phosphatase (Sigma) according to the manufacturer’s protocol. Plates were scanned using a CellCelector (Aviso) and scored manually.
accutase
Alkaline Phosphatase
Biological Assay
Cells
Embryonic Stem Cells
Gelatins
Glycogen Synthase Kinase 3
inhibitors
Laminin
PD-0325901
Serum
Stem Cells
2',5'-oligoadenylate
ABT-737
Annexin A5
Antibodies
Apoptosis
Biological Assay
Cell Lines
Cell Survival
Glycogen Synthase Kinase 3
Mass Spectrometry
Phosphorylation
Phosphotransferases
Plasmids
Proteins
RNA, Small Interfering
Sorafenib
Ubiquitination
Actins
Antibodies
FRAP1 protein, human
Glycogen Synthase Kinase 3
Ribosomal Protein S6 Kinases, 70-kDa
Tuberous Sclerosis 2
Antibodies
Cells
Collagenase
Digestion
dispase
Glycogen Synthase Kinase 3
Heparin
ITGA6 protein, human
Mammary Gland
Mus
Population Group
Serum
TACSTD1 protein, human
Trypsin
Y 27632
Most recents protocols related to «Glycogen Synthase Kinase 3»
hiPSCs were seeded into 10-cm tissue culture plates and then allowed to grow for ~4 days until the cells became ~70% confluent. CMs were differentiated from iPSCs as previously described (79 (link)) [protocol originally adapted from Lian et al. (35 (link))]. Media were changed to RPMI 1640 + B27 + ascorbic acid (RPMI/B27/AA) containing the GSK3 inhibitor CHIR99021 (Selleck Chemicals) (6 μM) on day 0 of CM differentiation. On day 2 of differentiation, media were changed to RPMI/B27/AA containing the Wnt antagonist IWP-4 (Reprocell) (5 μM). On day 4 of differentiation, media were changed to fresh RPMI/B27/AA, and on day 7 of differentiation, basal media were changed to RPMI/B27 + insulin (10 μg/ml), with media subsequently changed every 2 to 3 days with RPMI/B27/insulin. Beating of CMs was usually observed between days 7 and 9 of differentiation. CMs were dissociated using the STEMdiff Cardiomyocyte Dissociation Kit (StemCell Technologies) and replated onto Geltrex-coated plates for further assay.
Ascorbic Acid
Biological Assay
Cells
Chir 99021
Glycogen Synthase Kinase 3
Human Induced Pluripotent Stem Cells
Induced Pluripotent Stem Cells
Insulin
Melia azedarach
Myocytes, Cardiac
Stem Cells
Tissues
For BioID immunoblots of N-term
BirA*-FLAG fusions, the respective
HeLa Flp-In T-REx stable pools were incubated with tetracycline (1
μg/mL) and biotin (50 μM) for 24 h, then washed once with
ice-cold PBS, harvested, and frozen at −80 °C. Cell pellets
were lysed at a 4:1 lysis buffer-to-pellet ratio (v/w) (50 mM Tris
pH 7.5; 150 mM NaCl; 0.4% SDS, 1% NP-40, 1.5 mM MgCl2,
1 mM EGTA, benzonase nuclease 1:1000, and Sigma-Aldrich protease inhibitor
cocktail, P8340, 1:500) by pipetting ∼10 times, sonicating
3 × 5 s at 50% amplitude, and then by freeze/thaw (dry ice to
37 °C). Samples were then rotated end-over-end for 30 min at
4 °C and then centrifuged for 20 min at 16,000g at 4 °C. Supernatants were collected and incubated with 25
μL (bead slurry) of prewashed streptavidin–Sepharose
6 (GE) with rotation overnight at 4 °C. Beads were collected
(500g for 2 min), the supernatant was discarded,
and the beads were transferred to new tubes in 500 μL of lysis
buffer. Beads were washed once with SDS wash buffer (50 mM Tris–HCl,
pH 7.5, 2% SDS) and twice with lysis buffer. A fraction of each protein
extract (Input) was saved before the incubation with the beads. Samples
(Input and IP) were prepared for SDS-PAGE by incubating for 15 min
at room temperature (RT) in 2× Laemmli buffer with 5 mM biotin
and then boiling for 15 min. The proteins were transferred to poly(vinylidene
difluoride) (PVDF) membranes (Immobilon-FL, Millipore) and probed
with antibodies to detect the FLAG-BirA* fusions and endogenous proteins:
FLAG M2 (1:1000, Sigma F3165), GSK-3 (1:1000, Sigma 05-412), hPRUNE
(1:1000, Sigma ABC98), MISP (1:1000, Sigma HPA049511), DCP1B (1:1000,
CST13233). Blots were imaged on an Odyssey CLx (LICOR) and analyzed
using Image Studio and Empiria Studio software.
BirA*-FLAG fusions, the respective
HeLa Flp-In T-REx stable pools were incubated with tetracycline (1
μg/mL) and biotin (50 μM) for 24 h, then washed once with
ice-cold PBS, harvested, and frozen at −80 °C. Cell pellets
were lysed at a 4:1 lysis buffer-to-pellet ratio (v/w) (50 mM Tris
pH 7.5; 150 mM NaCl; 0.4% SDS, 1% NP-40, 1.5 mM MgCl2,
1 mM EGTA, benzonase nuclease 1:1000, and Sigma-Aldrich protease inhibitor
cocktail, P8340, 1:500) by pipetting ∼10 times, sonicating
3 × 5 s at 50% amplitude, and then by freeze/thaw (dry ice to
37 °C). Samples were then rotated end-over-end for 30 min at
4 °C and then centrifuged for 20 min at 16,000g at 4 °C. Supernatants were collected and incubated with 25
μL (bead slurry) of prewashed streptavidin–Sepharose
6 (GE) with rotation overnight at 4 °C. Beads were collected
(500g for 2 min), the supernatant was discarded,
and the beads were transferred to new tubes in 500 μL of lysis
buffer. Beads were washed once with SDS wash buffer (50 mM Tris–HCl,
pH 7.5, 2% SDS) and twice with lysis buffer. A fraction of each protein
extract (Input) was saved before the incubation with the beads. Samples
(Input and IP) were prepared for SDS-PAGE by incubating for 15 min
at room temperature (RT) in 2× Laemmli buffer with 5 mM biotin
and then boiling for 15 min. The proteins were transferred to poly(vinylidene
difluoride) (PVDF) membranes (Immobilon-FL, Millipore) and probed
with antibodies to detect the FLAG-BirA* fusions and endogenous proteins:
FLAG M2 (1:1000, Sigma F3165), GSK-3 (1:1000, Sigma 05-412), hPRUNE
(1:1000, Sigma ABC98), MISP (1:1000, Sigma HPA049511), DCP1B (1:1000,
CST13233). Blots were imaged on an Odyssey CLx (LICOR) and analyzed
using Image Studio and Empiria Studio software.
Antibodies
Benzonase
Biotin
Buffers
Cells
Cold Temperature
Dry Ice
Egtazic Acid
Freezing
Glycogen Synthase Kinase 3
Immobilon
Immunoblotting
Intestinal Atresia, Multiple
Laemmli buffer
Magnesium Chloride
Nonidet P-40
Peptide Hydrolases
Poly A
polyvinylidene fluoride
Proteins
SDS-PAGE
Sodium Chloride
Streptavidin
Tetracycline
Tissue, Membrane
Tromethamine
X. tropicalis embryos were injected with GSK3 ciliary biosensor (100 pg per embryo) and cultured until St. 8 and animal cap explants were dissected. Eight explants were seeded with the apical side on Lab-Tek II 4-well chambers coated with 1 mg/ml Concanavalin A (Sigma). The explants were cultured in Steinberg’s Solution until St. 30. Imaging was performed on an LSM780 confocal microscope (Zeiss). Z-stacks were taken at 0.38 µm steps at 5 min time intervals for a total of 30 time points. After the 6th time point, at 0:27, 2 µg/ml WNT3A recombinant protein or control buffer (0.1% BSA, 0.1 mM EDTA, 0.5% (w/v) CHAPS, 0.5% (w/v) DMSO in PBS) were added.
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3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate
Animals
Biosensors
Buffers
Concanavalin A
Edetic Acid
Embryo
Glycogen Synthase Kinase 3
Microscopy, Confocal
Sulfoxide, Dimethyl
Wnt3A Protein
A Zeiss LSM 700 microscope was used for imaging of IF samples. The pinhole was set to 1 Airy unit at maximal optical resolution before gain and offset were calibrated. Fluorescent signal intensities were within the linear range of detection. For all IF image analyses, ImageJ software was used. For high resolution IF, a 40x silicone immersion objective (numerical aperture: 1.25) was used at the Nikon AX confocal microscope. Pixel size was set to 100 nm and z sections were obtained at the ventral rim of the embryo to reduce background signal. Images were deconvolved using Nis-Elements 3D Deconvolution. Quantification of the ciliogenesis defects in X. tropicalis MCC cells was performed by immunostaining against acetylated tubulin and phalloidin to identify the ciliary axoneme and MCC border, respectively. MCC motile cilia were classified as ‘Normal’ or ‘Defect’ (showing lower cilia density and shorter cilia) as well as ‘Mild’ (>half the length compared to control), ‘Severe’ (X. tropicalis embryos were examined by calculating the ratio of MCC or Ionocytes to total cells. pLrp6 stainings as well as LRP6 WT, LRP6VA and LRP6(VA)P(PA) stainings were quantified by measuring their IF-positive areas and then normalizing to the AcTub+ area for each MCC using Fiji (ImageJ). Each data point represents one ratio. For the GSK3 ciliary biosensor live cell imaging, the mean signal intensity was measured for one MCC per experiment and time-point. The mean signal intensity for each MCC was normalized to that of the first time point (t = 0) in the representative graph. In fixed ciliary GSK3 biosensor-injected embryos, one full image per embryo was measured using Fiji (ImageJ). Ciliary GSK3 mean intensity was normalized to Arl13b-mKate2 mean intensity in the latter.
Images for phenotypical analyses were taken with the AxioCam MRc 5 microscope (Zeiss). Embryos were scored blind and data are representative images from 2 or more independent experiments. Representative embryos were selected using Magnetic Lasso tool of Adobe Photoshop CS6. Background was adjusted uniformly for presentation. Phenotypical features were analyzed by comparison to control embryos and classified and ‘normal’ and ‘defect’ (showing DV/AP patterning defects).
Images for phenotypical analyses were taken with the AxioCam MRc 5 microscope (Zeiss). Embryos were scored blind and data are representative images from 2 or more independent experiments. Representative embryos were selected using Magnetic Lasso tool of Adobe Photoshop CS6. Background was adjusted uniformly for presentation. Phenotypical features were analyzed by comparison to control embryos and classified and ‘normal’ and ‘defect’ (showing DV/AP patterning defects).
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Axoneme
Biosensors
Blindness
Cells
Cilia
Embryo
Eyelashes
GART protein, human
Glycogen Synthase Kinase 3
LRP6 protein, human
Microscopy
Microscopy, Confocal
Motile Cilia
Phalloidine
Silicones
Staining
Submersion
Tubulin
Vision
Mutagenesis of LRP6VA and LRP6(VA)P(PA) was performed by PCR-amplifying full length LRP6 with PhusionTM DNA polymerase using primers designed to mutate selected amino acids into alanines: LRP6VA forward: 5’-GATTCAGAACCTGCGCCCCCACCTCCCACACCC-3’; LRP6(VA)P(PA) forward: 5’-GATTCAGAACCTGCGCCCGCACCTCCCACACCC-3’; common reverse: 5’-GGGTGTGGGAGGTGCGGGCGCAGGTTCTGAATC-3’. GoTaq (Promega; Cat# M7841) DNA polymerase was used to amplify full-length Ppp1r11 using cDNA obtained from whole X. tropicalis embryos (Forward: 5’-TAAGCAGAATTCATGGCAGAATCCTCCGGG-3’; Reverse: 5’-TGCTTACTCGAGTTAGTGTTGCATGCTGCC-3’). Where indicated, N-terminal Flag tags were added via forward primers (Forward: 5’-TAAGCAGAATTCATGGATTACAAGGATGACGACGATAAGGCAGAATCCTCCGGGCCG-3’). The GSK3 ciliary biosensor (pArl13b-GSK3) was generated as follows: mouse Arl13b CDS without stop codon and the coding sequence of a linker peptide (Glycine-Glycine-Glycine-Glycine-Serine) were inserted upstream of the GFP coding sequence in a pCS2-GFP-GSK3-MAPK-Flag backbone plasmid (kind gift from De Robertis lab, see ref. 39 (link)). The mArl13b-linker part was amplified from E13.5 mouse whole brain cDNA by using the forward primer 5’-TTGCAGGATCCGCCACCATGTTCAGTCTGATGGCCAACTG-3’, and the reverse primer 5’-CTTGCTCACTGATCCTCCTCCTCCTGAGATCGTGTCCTGAGCAT-3’. The Linker-pCS2-GFP-GSK3-MAPK-Flag part was amplified from the pCS2-GFP-GSK3-MAPK-Flag backbone with the forward primer 5’-CGATCTCAGGAGGAGGAGGATCAGTGAGCAAGGGCGAGGAGCT-3’ and the reverse primer 5’-CAGTTGGCCATCAGACTGAACATGGTGGCGGATCCTGCAA-3’. Gibson assembly was performed according to the protocol from the NEB Gibson assembly kit (#E5510). Positive clones were picked and confirmed by Sanger-sequencing.
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Alanine
Amino Acids
Biosensors
Brain
Clone Cells
Codon, Terminator
DNA, Complementary
DNA-Directed DNA Polymerase
Embryo
Glycine
Glycogen Synthase Kinase 3
LRP6 protein, human
Mice, House
Mutagenesis
Oligonucleotide Primers
Open Reading Frames
Peptides
Plasmids
Promega
Serine
Vertebral Column
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LiCl is a chemical compound consisting of lithium and chlorine. It is a crystalline solid that is highly soluble in water and other polar solvents. LiCl is commonly used as a laboratory reagent and in various industrial applications.
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CHIR99021 is a small molecule compound that functions as a glycogen synthase kinase-3 (GSK-3) inhibitor. It is commonly used in cell culture and stem cell research applications.
More about "Glycogen Synthase Kinase 3"
Glycogen Synthase Kinase 3 (GSK-3) is a crucial serine/threonine protein kinase that plays a pivotal role in regulating glycogen metabolism, cell signaling, and development.
This enzyme is responsible for the phosphorylation and inactivation of glycogen synthase, a rate-limiting enzyme in the process of glycogen synthesis.
GSK-3 has been implicated in the pathogenesis of various diseases, including diabetes, neurodegenerative disorders, and cancer.
Understanding the intricate function and regulation of GSK-3 is essential for the development of targeted therapies.
Researchers often utilize compounds like LiCl, CHIR99021, and DMSO to investigate the role of GSK-3 in cellular processes.
Culturing cells in media containing FBS, Penicillin/Streptomycin, GlutaMAX, L-glutamine, DMEM/F12, and Non-essential amino acids can provide an optimal environment for studying GSK-3 and its interactions.
By leveraging the insights gained from these culture conditions and the latest advancements in AI-driven protocol comparisons, scientists can uncover reproducible and accurate findings from literature, pre-prints, and patents, ultimately identifying the best protocols and products for their GSK-3 research needs.
This enzyme is responsible for the phosphorylation and inactivation of glycogen synthase, a rate-limiting enzyme in the process of glycogen synthesis.
GSK-3 has been implicated in the pathogenesis of various diseases, including diabetes, neurodegenerative disorders, and cancer.
Understanding the intricate function and regulation of GSK-3 is essential for the development of targeted therapies.
Researchers often utilize compounds like LiCl, CHIR99021, and DMSO to investigate the role of GSK-3 in cellular processes.
Culturing cells in media containing FBS, Penicillin/Streptomycin, GlutaMAX, L-glutamine, DMEM/F12, and Non-essential amino acids can provide an optimal environment for studying GSK-3 and its interactions.
By leveraging the insights gained from these culture conditions and the latest advancements in AI-driven protocol comparisons, scientists can uncover reproducible and accurate findings from literature, pre-prints, and patents, ultimately identifying the best protocols and products for their GSK-3 research needs.