Figure 6

Effect of ganetespib on the maturation of the DYRK1A-DYRK1B chimera in mammalian cells. HEK293 cells transiently expressing DYRK1A, DYRK1B or chimeric constructs with an N-terminal FLAG tag and a C-terminal HiBiT tag were treated with 100 nM ganetespib (Gan) for 24 h or were not treated. Cells were extracted using a non-denaturing lysis buffer, and detergent-soluble fractions and insoluble fractions were separated by centrifugation. (A,B) Tyrosine autophosphorylation. The relative phosphotyrosine content of DYRK constructs in the soluble fraction was assayed by anti-FLAG immunoprecipitation and immunoblot analysis. HiBiT-containing proteins were detected using a fragment complementation assay (HiBiT blotting detection system). The graph shows means and SD of 3 experiments (B). (C,D) Accumulation in the insoluble fraction. Igepal-insoluble proteins in the pellets were solubilized in SDS buffer and DYRK1 constructs were quantitatively using the HiBiT blotting detection system (C). The concentration of HiBiT-tagged proteins in the soluble fraction was measured using the HiBiT lytic detection system. For each construct, the relative proportion of insoluble protein was calculated by dividing the HiBiT signals in the pellet by the HiBiT lytic values for the soluble protein as obtained in the respective supernatants (panel D; means and SD, n = 3). Statistical significance is indicated only for the treatment effects (*p < 0.05, **p < 0.01, ***p < 0.001).