Rabbit anti yap1

PD173074 was purchased from Sigma and used at 1 μm. FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml. YAP1-specific siRNA was purchased from Dharmacon, and the siRNAs targeting LAST1 or LAST2 were purchased from Sigma Aldrich. The following primary antibodies were used: rabbit-anti-E-cadherin (Abcam), rabbit-anti-Snail (Abcam), mouse-anti-Vimentin (Santa Cruz, Abcam), mouse-anti-FGF8 (Abcam), rabbit-anti-YAP1 (Abcam), rabbit-anti-LATS1 (Abcam), rabbit-anti-LATS2 (Abcam), rabbit-anti-Histone H3 (Abcam).

HOC1a cells were treated with 10 μM gossypol for 4 h. The control and gossypol-treated cells were lysed with RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, protease inhibitors, and phosphatase inhibitors l from Solabio (Beijing, China)) for 30 min on ice. The supernatant was collected after centrifugation for 30 min at 4°C. Protein concentrations were determined using a BCA assay. Proteins were separated by 1D SDS-PAGE and transferred onto a PVDF membrane by electroblotting. After blocking with 5% nonfat milk for 2 h at room temperature, the membrane was incubated overnight at 4°C with 2000-fold diluted primary antibody (rabbit anti-YAP1, from Abcam, UK), washed with TBST buffer for 3 times (each 10 minutes), and then incubated for 2 h with 2000-fold diluted anti-rabbit secondary antibody labeled with HRP. After washing with TBST buffer for 3 times (each 10 min), images were developed using Enlight western blotting detection reagents from Engreen Biosystem (Beijing, China). β-Actin was detected with anti-β-actin antibodies as an internal control.