Ab109001

The total protein of tissues and cells was extracted. After ice bathing and centrifugation, the sample was added to each lane for electrophoresis separation. The separated protein was transferred onto a nitrocellulose membrane. Then, the nitrocellulose membrane was blocked with 5% skim milk powder at 4°C overnight and incubated overnight with primary rabbit anti‐mouse polyclonal antibodies diluted at the ratio of 1:1000: NEK2 (ab115731), β‐catenin (ab16051), Nanog (ab21624), Oct‐4 (ab19857), CXCL16 (ab101404), EGFR (ab52894), TCF‐4 (ab217668) and Pygo2 (ab109001) and rabbit anti‐mouse monoclonal antibodies diluted at 1:1000: CD24 (ab64064), and CD44 (ab51037), all of which were purchased from Abcam Inc, Cambridge, UK. The membrane was washed 3 times with PBS at room temperature, 5 minutes for each, incubated with oscillation with the addition of the secondary antibody of horseradish peroxidase (HRP)‐labelled goat anti‐rabbit immunoglobulin (IgG) (1:10 000, ab6728, Boster Biological Technology Co. Ltd, Wuhan, China) at 37°C for 1 hour, washed with Tris‐buffered saline‐Tween (TBST) and visualized using HRP electroluminescence (ECL). Grey value analysis of the target bands was performed using ImageJ software, and the experiment was repeated three times independently.

Tissues were fixed with 10% neutral formalin, embedded in paraffin, and 3-μm-thick sections were prepared by pathological technologist. Hematoxylin-eosin (HE) stain was performed as previous describe [17 ]. For immunohistochemistry (IHC) staining, sections were deparaffinized, hydrated and soaked in 3% H₂O₂ for 15 minutes at room temperature, and then incubated with Pygo2 polyclonal antibody (1:4000, ab109001, Abcam) and E-cadherin (1:1000, 20874-1-AP, Proteintech) at 4°C overnight. Biotinylated secondary antibody and diaminobenzidine were purchased from Maixin Biotechnology (Fuzhou, China). Evaluation of Pygo2 and E-cadherin staining in HCC tissue sections was performed based on the IHC assessment methods used by Popadiuk et al. for epithelial ovarian cancer [12 (link)] and Motoyuki Hashiguchi et al. for HCC [44 (link)], respectively.

Total protein was extracted with RIPA lysis buffer supplemented with Protease Inhibitor Cocktail (Sigma, St Louis, MO, USA) and quantified using the Bradford method. Protein lysate was loaded on 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to the polyvinylidene fluoride membrane. After blocking the membranes with non-fat milk, the blots were incubated with primary antibodies directly against Pygo2 (1:1000, ab109001, Abcam), β-Actin (1:1000, Cell signaling technology), E-cadherin (1:800, 20874-1-AP, Proteintech), Zeb2 (1:500, 14026-1-AP, Proteintech), Fibronectin (1:1000, Cell signaling technology), N-cadherin (1:1000, Cell signaling technology), MMP-2 (1:1000, Cell signaling technology) at 4°C overnight. After that, the blots were incubated with the secondary antibody labeled with horse radish peroxidase at room temperature for 2 h. Protein bands were visualized using enhanced chemiluminescence and quantitated by densitometry using Image-J software. The relative protein levels were calculated by comparison to the amount of β-Actin protein. Experiments were repeated in triplicate.