Fig. 3.

Trametinib downregulates YAP via inhibiting MEK nuclear localization. a Western blotting for YAP and CYR61 in MDA-MB-231 cells treated with increasing concentration of trametinib (50–400 nM). b WB for YAP and CYR61 using MDA-MB-231, SW480 and SW1116 cells treated with LMB (10 ng/ml, 4 h) or left untreated in the presence or absence of trametinib (100 nM, 24 h). c Extracting the cytoplasmic and nuclear protein, then WB for MEK and β-TrCP in SW1116 and SW480 cells treated with LMB (10 ng/ml, 4 h) or left untreated in the presence or absence of trametinib (100 nM, 24 h). d Quantitative real-time RT–PCR to measure YAP and CYR61 mRNA levels in SW1116 cells treated with trametinib (100 nM, 24 h) or DMSO. GAPDH was used as a control. ***p < 0.001 using Student’s t test (two-tailed). e WB for YAP and CYR61 using SW1116 and SW480 cells treated with trametinib (100 nM, 24 h) or DMSO supplemented with or without MG132 (20 μM) for 10 h. f WB for YAP, CYR61 and β-TrCP using SW1116 and SW480 cells treated with trametinib (100 nM, 24 h) or DMSO combined with or without siTrCP. g SW1116 cells were transfected with FLAG-YAP and HA-β-TrCP, then treated with trametinib (100 nM, 24 h) or left untreated. MG132 (20 μM, 10 h) was used to prevent YAP degradation. Anti-TrCP antibody was used for IP. Blots were probed with anti-TrCP and anti-YAP