Anti gli1

RIPA buffer was used to extract total protein from the palatal explants. A BCA Protein Assay Kit (Solarbio, China) was then used to detect protein content in the supernatant. The protein was denatured at 100 °C for 5 min, then (∼20 μg) electrophoresed on SDS-PAGE gel and transferred onto a PVDF membrane (Merck Millipore, USA). The membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 and incubated with primary antibodies at 4 °C overnight. The antibodies used were anti-Laminin α5 (Abcam, AB184330, 1:1000), anti-ki67 (Affinity, AF0931, 1:1000), anti-cyclin D1 (Affinity, AF0931, 1:1000), anti-cleaved caspase 3 (Cell Signaling Technology, 9664, 1:1000), anti-gli1 (Affinity, AF0931, 1:1000), anti-β-actin (Elabscience, E-AB-20058, 1:1000). Then the membranes were incubated with a secondary antibody at room temperature for 1.5 h. An ECL kit (Cell Signaling Technology, USA) was used to detect the protein bands following the manufacturer's protocols. β-actin was used as the load control. The grey level was quantified using ImageJ v1.8.0 software (National Institutes of Health).