For in vitro AKT1 kinase activity assays, recombinant active AKT1 kinase (Abcam) and GSK-3β (SinoBiological) substrates were added to 30 μl kinase buffer. The AKT1 kinase activity was indicated by the phosphorylation of GSK-3β. Kinase reactions were performed at 30°C for 30 min, and the samples were heated to 100°C for 5 min followed by the addition of SDS loading buffer. Reaction products were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Then Total-GSK-3β (T-GSK-3β) (Proteintech), phosphorylated GSK-3β (P-GSK-3β) (Proteintech) and Total-AKT1 (T-AKT1) proteins were determined by Western blotting.
Gsk3β
Manufactured by Sino Biological
Sourced in China
GSK3β is a protein kinase that plays a key role in the regulation of various cellular processes, including metabolism, cell survival, and cell structure. It is a member of the glycogen synthase kinase-3 (GSK3) family of enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Ab89621, by Abcam (1 mentions)
Ab185655, by Abcam (1 mentions)
Ubiquitin, by Proteintech (1 mentions)
Ab185656, by Abcam (1 mentions)
Ab140601, by Abcam (1 mentions)
Dab2ip, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ab32072, by Abcam (1 mentions)
Superdex 75 10 300 gl, by GE Healthcare (1 mentions)
4 protocols using gsk3β
1
AKT1 Kinase Activity Assay
2
Comprehensive Immunoblotting Assay Protocol
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Immunoblotting assays were carried out as previously described9 with the following primary Abs: DAB2IP (23582‐1‐AP, Proteintech), c‐Myc (ab32072, Abcam), c‐Myc (67447‐1‐Ig, Proteintech), p‐c‐MycS62 (ab185656, Abcam), p‐c‐MycT58 (ab185655, Abcam), glycogen synthase kinase 3β (GSK3β; 10044‐T32, SinoBiological), p‐GSK3βS9 (#5558, CST), Bcl‐2 (12789‐1‐AP, Proteintech), Bax (#5023, CST), B56α (ab89621, Abcam), Ubiquitin (10201‐2‐AP, Proteintech), Ubiquitin (linkage‐specific K48) (ab140601, Abcam), Nanog (#8822S, CST), CD44 (60224‐1‐Ig, Proteintech), CD133 (18470‐1‐AP, Proteintech), and GAPDH (60004‐1‐Ig, Proteintech). Secondary Abs conjugated to HRP (Servicebio) were used, followed by chemiluminescence.
3
Phosphorylation of Tau by GSK3β
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Purified Tau was incubated with GSK3β (SinoBiological) in a 100:1 ratio, using a buffer of 25 mM HEPES pH 7.5, containing 100 mM NaCl, 10 mM MgCl2, 10 mM ATP, and 2 mM TCEP. The reaction was allowed to proceed for 19 hours at 37 °C. Verification that the reaction had gone to completion was performed by SDS-PAGE with Coomassie blue staining. The pTau was further purified by size exclusion chromatography using a Superdex 75 10/300 GL (GE Healthcare) column in buffer consisting of 20 mM Tris-HCl pH 8, 200 mM NaCl, and 2mM TCEP. The concentration of purified pTau was determined by UV absorbance at 280 nm. Fractions were collected and frozen in −80 prior to use.
4
Calorimetric Study of AB4 Binding to GSK-3β
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ITC experiment on the interaction of AB4 with GSK‐3β (50650‐M07B, SinoBiological, China) were carried out at 25°C using an iTC200 titration calorimetry (MicroCal, Northampton, MA). GSK‐3β was diluted in 20 mM Tris‐HCl buffer (pH 7.4) at 10 μM, and loaded into the sample cell (200 μL). A solution of 100 μM AB4 was placed in the injection syringe (40 μL). The ITC titration data are fitted to a single set of identical sites model using the MicroCal ORIGIN software supplied with the instrument.
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