Figure 2

Temperature dependence of GST-DYRK1B maturation and stability. (A,B) Temperature effects on maturation of the catalytic domain. GST-DYRK1AΔC and GST-DYRK1BΔC were expressed in E. coli at 37 °C (4 h), 19 °C (23 h) or 8 °C (48 h). GST fusion proteins were partially purified by affinity adsorption to glutathione Sepharose before Western blot analysis (A). Panel (B) shows the quantitative evaluation of n = 3 experiments (means and SD). (C) Thermal stability of the mature kinase domain. Samples of GST-DYRK1AΔC and GST-DYRK1BΔC with similar catalytic activity were heat challenged for 3.5 min at variable temperatures before the proteins were subjected to a kinase assay with a peptide substrate. Catalytic activities were normalized to the activity of a control sample that was not exposed to heat shock. This result was reproduced with independent preparations of GST-DYRK1A and GST-DYRK1B (supplementary Fig. S2). AU arbitrary units.