Ecl substrate solution kit

Western blots were performed to determine the protein expression levels of CD24, CD133 and EpCAM in the liver tissues. Briefly, protein lysates were extracted from the liver tissues using RIPA Lysis Buffer (Aspen, Canada, USA) supplemented with the protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentration was determined by BCA protein kit (Aspen, Canada, USA). The protein samples were separated with 10% SDS-PAGE and transferred to PVDF membrane. Then, the membrane were incubated with the primary antibodies overnight at 4 °C, followed by the corresponding horseradish peroxidase conjugated secondary antibodies at room temperature for 30 min. Finally, the membrane was developed with the enhanced chemiluminescence (ECL) substrate solution kit (Aspen, Canada, USA) for 1 min. The targeted protein bands were analyzed using with AlphaEaseFC software for gray scale value. GAPDH was used as the internal control. Primary antibodies used in this study are listed in Table 2.Table 2

Detailed information for antibodies used for western blot

Antibody	Species	Manufacturer	Category no	Dilution ratio
GAPDH	Rabbit	Abcam	ab9485	1:10,000
CD24	Rabbit	Abcam	ab179821	1:500
CD133	Rabbit	Cell Signaling Technology	64,326	1:500
EpCAM	Rabbit	Abcam	ab223582	1:1000
Wnt3a	Rabbit	Abcam	ab219412	1:500
β-catenin	Rabbit	Abcam	ab32572	1:500