Cultured cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer. Protein concentrations were determined using a micro bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Twenty micrograms total protein extract was separated by 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and electroblotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membrane was then blocked and then incubated with primary antibodies overnight at 4°C.
The primary antibodies used included the followings: anti-Col1a2, anti-Col3a1, anti-α-SMA (Abcam; 1:1000), anti-cyclin-dependent kinase (CDK) family, anti-cyclin D1, anti-cyclin E1, anti-smads, anti-MAPK family (Cell Signaling Technology, Beverly, MA, 1:1000), anti-GAPDH (Sigma-Aldrich; 1:10,000). Immunoreactive bands were quantitatively analyzed with ImageJ software.
The primary antibodies used included the followings: anti-Col1a2, anti-Col3a1, anti-α-SMA (Abcam; 1:1000), anti-cyclin-dependent kinase (CDK) family, anti-cyclin D1, anti-cyclin E1, anti-smads, anti-MAPK family (Cell Signaling Technology, Beverly, MA, 1:1000), anti-GAPDH (Sigma-Aldrich; 1:10,000). Immunoreactive bands were quantitatively analyzed with ImageJ software.