Smoothened

For cell staining, Ptch^−/− MEF cells were cultured and seeded on coverslips. Then, 4% (w/v) paraformaldehyde/phosphate-buffered saline (PBS) was used to fix cells at room temperature for 20 min. After fixation, the cells were washed three times with PBS, blocked with 5% (w/v) bovine serum albumin (BSA)/PBS at room temperature for 1 h, and incubated with a primary antibody in 5% BSA overnight at 4 °C. Then, the cells were washed and treated with secondary antibodies conjugated with Alexa Fluor 488 or 568 dyes in 5% BSA at room temperature for 1 h. The images were achieved through LSM710 Zeiss confocal microscope and analyzed with Image J software. The following primary antibodies were applied for immunostaining: ARL 13B (Proteintech, Wuhan, China, 17711-1-AP, 1:100); and Smoothened (Santa Cruz, CA, USA, sc-166685, 1:100).

For tissue staining, dorsal skin were fixed with 4% paraformaldehyde, dehydrated in graded ethanol series and embedded in paraffin. Sections of 5 µm thickness were prepared by Leica RM2255 rotary microtome and used for immunostaining after deparaffinization. For cell staining, Ptch^−/− MEF cells cultured on cover-slips were fixed with 4% paraformaldehyde, blocked with 5% BSA for 1 h and incubated overnight with primary antibody. The following antibodies were used for immunostaining: Smoothened (Santa Cruz, sc-166685, 1:100); ARL 13B (Proteintech, 17711-1-AP, 1:100); Gli1 (Proteintech, 66905–1-Ig, 1:200). The secondary antibodies conjugated with Alexa Fluor 488 or 568 were purchased from Invitrogen, those conjugated with horse radish peroxidase (HRP) using DAB as chromogen from Cwbio. Sections were photographed by LSM710 Zeiss confocal microscope and analyzed with Image J software.