Fig. 4
Domperidone inhibits MEK/ERK pathway and CDK4/SMAD3 pathway
(A) MEK1 and MEK2 (B) kinase activity was assessed by an in vitro kinase assay using p-ERKT185/Y187 antibody. Active MEK1 or MEK2 incubated with inactive ERK2 in kinase reaction buffer together with different dose of domperidone and then followed by Western blot analysis. (C) KYSE150 cells were co-transfected with CDK4-Flag and CylinD1-HA, and then treated with 40 µM or 60 µM domperidone for 24 h. Cell lysates were immunoblotted with anti-p-SmadT8 antibody. Mutation version MEK1 (D) or MEK2 (E) incubated with inactive ERK2 in kinase reaction buffer, followed by Western blot analysis. (F) KYSE150 cells were co-transfected with either CDK4-Flag or CDK4 K35A-Flag along with CylinD1-HA. Cell lysates were immunoblotted with anti-p-SMADT8 antibody. (G) KYSE150 and KYSE450 cells were treated with different concentration of domperidone after 24 h. Protein levels of P-ERK, p-MEK1/2, T-ERK, T-MEK1/2, p-SMAD3, T-SMAD3, CDK4, P21 and c-myc were analyzed by Western blot. (H) Pull down assay was used to evaluate the binding affinity between domperidone and c-myc or P21. (I) KYSE150 cells and CDK4 knock out cells were treated with 40 µM domperidone for 24 h, and then Western blot was performed. (J) KYSE150 was incubated with 40 µM domperidone or domperidone and SMAD3 inhibitor (E)-SIS3 3µM for 24 h simultaneously.