The cytosolic and nuclear fractions of cells were prepared as previously described (Wu et al. 2008 (link); Gong et al. 2014 (link)). Immunoprecipitation and western blot analyses were performed using standard protocols as previously described (Wu et al. 2008 (link)). The in vitro kinase assay to assess the phosphorylation of GSK3β by JAK2 was performed as previously described (Wu et al. 2008 (link)). To validate JAK2 as a kinase phosphorylating GSK3β at Y216, recombinant human active JAK2 (Abcam, ab42621) and human GSK3β (Abcam, ab63193) were used in the in vitro kinase assay. 30 ng of GSK3β was incubated with 50 μl kinase assay buffer (Cell Signaling) in the presence or absence of 30 ng active JAK2 and 2 mM ATP at 37 °C for 30 min. The reactive mixture was subjected to western analyses with antibodies recognizing p-Tyr216-GSK3β, GSK3β, and JAK2. For western, GAPDH or β-actin was used as the internal standard of total target proteins, and phosphorylated proteins were normalized to total proteins, respectively. Immunoreactive bands from duplicates or triplicates were quantified by ImageJ, and the mean intensity from first band was set to 1.
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