Cells were lysed in RIPA buffer (Sigma-Aldrich) consisting of 50 mmol/L
Tris-HCI, 1% Nonidet P40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L
ethylenediaminetetraacetic acid, 1 mmol/L activated
Na3VO4,1 mmol/L NaF, and 1X of Halt Protease/Phosphatase
Inhibitor Cocktail (Thermo Fisher Scientific). From the lysates, 15 μg of
protein was resolved by electrophoresis on a 4%-15% sodium dodecyl
sulfate-polyacrylamide gradient gel. After electrophoresis, the gel was
transferred to immunoblot polyvinylidene difluoride membrane (Bio-Rad). Western
blotting was performed by using antibodies against phospho (S127)-YAP (Cell
Signaling Technology), phospho (S89)-TAZ (Cell Signaling Technology), collagen I
antibody (Novus Biologicals), phospho(S133)-CREB (Abcam), phospho(Y357)-YAP
(Abcam) and GAPDH (Cell Signaling Technology) as internal control. The image was
developed by SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher
Scientific), and detected by Bio-Rad ChemiDoc imaging system (Bio-Rad). The band
intensity from the images was analyzed by Image Lab™ software
(Bio-Rad).
Tris-HCI, 1% Nonidet P40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L
ethylenediaminetetraacetic acid, 1 mmol/L activated
Na3VO4,1 mmol/L NaF, and 1X of Halt Protease/Phosphatase
Inhibitor Cocktail (Thermo Fisher Scientific). From the lysates, 15 μg of
protein was resolved by electrophoresis on a 4%-15% sodium dodecyl
sulfate-polyacrylamide gradient gel. After electrophoresis, the gel was
transferred to immunoblot polyvinylidene difluoride membrane (Bio-Rad). Western
blotting was performed by using antibodies against phospho (S127)-YAP (Cell
Signaling Technology), phospho (S89)-TAZ (Cell Signaling Technology), collagen I
antibody (Novus Biologicals), phospho(S133)-CREB (Abcam), phospho(Y357)-YAP
(Abcam) and GAPDH (Cell Signaling Technology) as internal control. The image was
developed by SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher
Scientific), and detected by Bio-Rad ChemiDoc imaging system (Bio-Rad). The band
intensity from the images was analyzed by Image Lab™ software
(Bio-Rad).