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. 2020 Jan 8;27(1):41–53.e6. doi: 10.1016/j.chom.2019.11.002

Figure 5.

Figure 5

SteE Enables GSK3 to Phosphorylate STAT3

(A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg His-MBP or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM ATP, were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats.

(B) GFP, WT GFP-GSK3α, and GFP-GSK3αL195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments.

(C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3αL195G; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.