Ab52189

Total proteins were extracted from tissues and cells. Anti-GAPDH antibody (1:5000, A5441, Sigma), anti-CRIF1 (1:2000, 16260-1-AP, Proteintech), anti-cyclin D1 (1:2000, ab137875, Abcam), anti-cyclin E1 (1:1000, ab52189, Abcam), anti-CDK4 (1:1000, ab137675, Abcam), anti-CDK6 (1:1000, ab151247, Abcam), anti-MMP3 (1:1000, ab52915, Abcam), anti-TGF-β1 (1:500, ab9758, Abcam), anti-TGF-β2 (1:100, ab167655, Abcam), TGF-β receptor 1 (1:1000, ab155258, Abcam), anti-Smad2 (1:1000, 3122, CST), anti-Smad3 (1:1000, 9513, CST), anti-p-Smad2 (1:1000, 3108, CST), anti-p-Smad3 (1:1000, 9520, CST), anti-E-Cadherin (1:2000, ab133597, Abcam), and anti-N-Cadherin (1:1000, ab19348, Abcam) antibodies were used. Immunodetection was performed using an EZ-ECL chemiluminescence detection kit (BeitHaemek, Israel).

Immunohistochemical staining for H₂A histone family, member X (γ-H₂AX, Abcam, ab81299), BrdU (Abcam, ab6326), Ki67 (Abcam, ab15580), Cyclin A2 (Abcam, ab181591), Cyclin E1 (Abcam, ab52189) and Cyclin D1 (Abcam, ab134175) were performed on 4% paraformaldehyde-fixed liver sections. 5μm-thick slices were incubated with primary antibodies at 4? overnight followed by biotinylated secondary antibody and the avidin/biotin horseradish peroxidase system (Vectastain DAB Kit; Vector Laboratories, Burlingame, CA). The nuclei were counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO) and the sections were covered in neutral balsam (Solarbio, Beijing, CHN). Immuno-fluorescent staining for β-catenin (Sigma-Aldrich, MAB2081) and PHH3 (Roche, 760-4591) were detected on paraffin-embedded liver section. Slides were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies conjugated with fluorescent dye at 37°C for 30 min.
Immunocytochemical staining for BrdU (Abcam, ab6326) and Ki67(Abcam, ab15580), cells were fixed, permeabilized and blocked, then incubated with primary antibody, followed by fluorescence-tagged secondary antibodies. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO). Images were acquired with a 50i Nikon fluorescence microscope (Nikon, Melville, NY).