Krt19

Total protein was extracted from the cells cultured in the plates using RIPA
Lysis Buffer (Beyotime Biotechnology, Shanghai, China). After being quantified
with a BCA protein quantification kit (Beyotime Biotechnology), 30 μg of total
proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and transferred to a PVDF transfer membrane (Millipore,
Shanghai, China). The membranes were then blocked with 5% skim milk in
Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 1 h and
hybridized with appropriate primary antibody (SOX9 (1 μg/mL; Abcam, Shanghai,
China), aggrecan (1 μg/mL; Abcam), collagen II (0.2 μg/mL; Abcam), collagen I (1
μg/mL; Abcam), KRT19 (1 μg/mL; Abcam), glypican 3 (2.5 μg/mL; Abcam), PAX1 (1.25
μg/mL; Abcam), GDF10 (10 μg/mL; Abcam) or GAPDH (0.1 μg/mL; Santa Cruz,
Shanghai, China)) at 4°C overnight. After that, the membranes were incubated
with a horseradish peroxidase-labeled secondary antibody (0.1 μg/mL; Santa Cruz)
for 1 h at room temperature. The immunoreactive bands were visualized using an
enhanced chemiluminescence substrate (Millipore). The expression levels of
protein were quantified by densitometry using the Image Lab™ Software (Bio-Rad
Laboratories Inc., Munich, Germany).

Total protein of each group was extracted by RIPA buffer containing 1% phenylmethanesulfonyl fluoride, and the concentrations were measured with a BCA quantification kit (Pierce, China). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 1 h, followed by incubating with appropriate primary antibodies overnight at 4°C in TBST: SOX9 (1:1,000; Abcam, UK), aggrecan (1:1,000; Abcam), collagen II (1:5,000; Abcam), KRT19 (1:1,000; Abcam), PAX1 (1:400; Abcam), GDF10 (1:400; Abcam), Shh (1:1,000; Cell Signaling Technology, China), PTCH1 (1:1,000; Cell Signaling Technology), Gli1 (1:1,000; Abcam), Gli2 (1:500; Abcam), Smad3 (1:1,000; Cell Signaling Technology), p-Smad3 (1:1,000; Cell Signaling Technology), or GAPDH (1:5,000; Santa Cruz, China). After that, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:5,000; Santa Cruz) at room temperature for 1 h, and immunoreactivity was detected using an enhanced chemiluminescence substrate (Millipore, China). The relative protein levels were quantified by densitometry using the Quantity One Software (Bio-Rad Laboratories, Germany).