T β cat

Cell-free GSK-3β activity assays were performed as previously described [34] (link). In brief, after IP, the HA-resin was washed twice with Tris-buffered saline with 0.1% Tween, followed by one wash with kinase reaction buffer (Cell Signaling Technology). Next, immunoprecipitated GSK3β (WT and mutant) was resuspended in 50 µL kinase reaction buffer in the presence of 1 μg β-catenin (β-Cat, Sigma-Aldrich) as substrate and 0.4 mM ATP (Cell signaling technology). To test the inhibitory effect of electrophiles, 4-HNE was added to the reaction system at the indicated concentrations for 1 h prior to initiation of the activity assay. The kinase reaction was carried out at 37 °C for 30 min under gentle agitation, and terminated by adding 4× reducing sample buffer (Bio-Rad), and denatured by heating at 100 °C for 5 min. The levels of phospho-β-catenin (p-β-Cat, S33/37/T41, Cell Signaling Technology) and total β-catenin (t-β-Cat, Cell Signaling Technology) were analyzed using Western blotting, and the ratios of p-β-Cat/t-β-Cat were calculated to represent GSK3β activity.