Fig. 4. Nuplazid inhibits the kinase activity of PAK4.
a PAK4 kinase activity was assessed by an in vitro kinase assay using active PAK4 and inactive MAPK1 proteins. The effect of Nuplazid was determined by western blot analysis using a p-MAPK1T185/Y187 antibody. b Nuplazid binds with PAK4 in an ATP-competitive manner. Active PAK4 was incubated with ATP at different concentrations (10, 20 or 100 µM) and 100 µl of Nuplazid–sepharose 4B or sepharose 4B (as a negative control) in reaction buffer. The level of p-PAK4, PAK4, p-MAPK1 and T-MAPK1 in KYSE150 cells (c) and KYSE450 cells (d) with different concentration of Nuplazid (0, 0.5, 1, 2.5 and 5 μM) treatment for 24 h was determined by western blotting. The level of p-MAPK1T185/Y187 was affected by PAK4 in KYSE150 cells (e) and KYSE450 cells (f) which treated with Nuplazid. MAPK1 was immunoprecipitated by PAK4 and MAPK1 was detected by p-MAPK1T185/Y187.