Medium 199 was obtained from Boehringer (Mannheim, Germany), fetal calf serum from PAA (Linz, Austria), crude collagenase from Biochrom (Berlin, Germany), and MMP inhibitors from Merck (Darmstadt, Germany). Antibodies for MMP-2, MMP-9, and MMP-14 and integrin1β were from Abcam, antibodies against ERK and p-ERK were from Santa Cruz (Heidelberg, Germany) and against p-GSK3β were from Cell Signaling (Frankfurt, Germany), and vinculin antibodies were from Sigma-Aldrich (Taufkirchen, Germany). Secondary antibodies were from Cell Signaling (Frankfurt, Germany). GSK-3β Activity Assay Kit was from Sigma.
Gsk 3β activity assay kit
Manufactured by Merck Group
Sourced in Canada, Spain
The GSK-3β Activity Assay Kit is a laboratory tool designed to measure the enzymatic activity of the glycogen synthase kinase-3 beta (GSK-3β) protein. GSK-3β is an important regulatory enzyme involved in various cellular processes. The kit provides the necessary reagents and protocols to quantify the activity of this enzyme in biological samples.
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Lab products found in correlation
Crude collagenase, by Harvard Bioscience (1 mentions)
Vinculin antibody, by Merck Group (1 mentions)
Medium 199, by Boehringer Ingelheim (1 mentions)
Scintillation counter, by Beckman Coulter (1 mentions)
Ezview red protein g affinity gel, by Merck Group (1 mentions)
G lisa rhoa activation assay biochem kit, by Cytoskeleton (1 mentions)
Anti c myc antibody, by Abcam (1 mentions)
5 protocols using gsk 3β activity assay kit
1
MMP and Integrin Signaling Assay
2
GSK-3β Activity Assay Protocol
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The GSK-3β activity was determined by the use of GSK3β activity assay kit from Sigma. The total protein was extracted as described for immunoblots. Then GSK3β was immunoprecipitated. After addition of a peptide substrate and γP32-ATP, labeling of the peptide by GSK-3β was determined.
3
Quantification of GSK-3β Activity
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GSK3β activity was measured using the GSK-3β activity assay kit (Sigma, ON, Canada) following the manufacturer’s protocol. Briefly, cells transfected with CDK5RAP2 or control siRNA were lysed in ice-cold lysis buffer containing 50 mM Tris/pH 8.0, 150 mM NaCl, 1% NP-40, 10 mM EDTA, 5% glycerol, 1 mM phenylmethylsulfonylflouride (PMSF), 10 µg/ml aprotinin and 10 µg/ml leupeptin. Cell lysates (300 µg) were incubated with 2 µl of anti-GSK-3β and 30 µl EZview Red protein-G affinity gel beads at 4 °C for 3 h. The beads were recovered by centrifugation at 8000 × g for 30 sec and washing with 500 µl of ice-cold lysis buffer. The immunoprecipitates were mixed with 20 µl of reaction mixture containing 25 μCi γ32P-ATP and 5 µl of GSK-3β substrate solution, incubated at 37 °C for 30 m, then spotted onto P81 phosphate cellulose membranes. Membranes were washed 4 times with 0.5% phosphoric acid and once with acetone. Counting of incorporated radioactivity was performed using a Beckman scintillation counter.
4
GSK-3β Phosphorylation Activity Assay
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The GSK-3β phosphorylation activity is determined by measuring the radioactivity of γ-phosphate radioactive residues introduced in the substrate by the GSK-3β enzyme using γ-adenosine triphosphate (ATP). GSK-3β activity was analyzed using GSK-3β Activity Assay Kit (CS0990; Sigma, Madrid, Spain), according to the producer’s procedures. GSK-3β was immunoprecipitated, in the lysed cells procured according to Moyano et al. [4 (link)], using a conjugate of agarose beads and anti-GSK-3β antibody (EZview Red Protein G Affinity Gel, P6486; Sigma, Madrid, Spain). Then, the GSK-3β obtained was incubated with substrate and γ32P-ATP, which introduces 32P residues into the substrate. The radioactivity emitted by the γ32P-substrate was quantified through scintillation counting and normalized with the absolute protein concentration as indicated above. Results obtained are presented as percentages of the untreated control.
5
ChIP-qRT-PCR Analysis of c-Myc Binding
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ChIP samples were prepared as previously reported 17 using a ChIP-tested anti-c-myc antibody (mouse clone 9E11; Abcam). The putative c-Myc binding site on ABCB5 promoter was validated with the Matinspector software (https://www.genomatix.de/matinspector.html). Primer sequences were: 5'-CACAACTTCAAGTGGTAGCATG-3'; 5'-CCATTCTACCCAGTGAAATG-3'. Primers used as negative internal controls for a non-specific 10000 bp upstream sequence were: 5'-GTGGTGCCTGAGGAAGAGAG-3'; 5'-GCAACAAGTAGGCACAAGCA-3′. The immunoprecipitated products were amplified by qRT-PCR.
GSK3β, RhoA and RhoA kinase activity. The kinase activity of GSK3β was measured on the protein immunopurified from cell extracts by a radiometric assay, using the GSK-3β Activity Assay Kit (Sigma Chemicals. Co), as per manufacturer's instructions. Results were expressed as count per minute (cpm)/mg cellular proteins. Rho-GTP, considered an index of active RhoA, and RhoA kinase activity, were measured by spectrophotometric methods, using the G-LISA RhoA Activation Assay Biochem Kit (Cytoskeleton, Denver, CO) and the CycLex Rho Kinase Assay Kit (CycLex, Nagano, Japan) respectively. Results were expressed U absorbance/mg cell proteins, according to titration curves prepared with serial dilutions of Rho-GTP positive control (Cytoskeleton) and recombinant RhoAK (MBL, Woburn, MA, USA).
GSK3β, RhoA and RhoA kinase activity. The kinase activity of GSK3β was measured on the protein immunopurified from cell extracts by a radiometric assay, using the GSK-3β Activity Assay Kit (Sigma Chemicals. Co), as per manufacturer's instructions. Results were expressed as count per minute (cpm)/mg cellular proteins. Rho-GTP, considered an index of active RhoA, and RhoA kinase activity, were measured by spectrophotometric methods, using the G-LISA RhoA Activation Assay Biochem Kit (Cytoskeleton, Denver, CO) and the CycLex Rho Kinase Assay Kit (CycLex, Nagano, Japan) respectively. Results were expressed U absorbance/mg cell proteins, according to titration curves prepared with serial dilutions of Rho-GTP positive control (Cytoskeleton) and recombinant RhoAK (MBL, Woburn, MA, USA).
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