Ab39266

Cells were seeded on cover slips in 12-well plates and allowed to grow to 80% confluence. Cells were washed twice with PBS and fixed 10 min with 4% paraformaldehyde (PFA, Sigma) and then incubated in PBS supplemented with 0.1% Tween and 1% BSA for 1 h to permeabilize the cells and block non-specific protein-protein interaction. Cells were then incubated with anti-Patched antibody (Ab39266 from Abcam 1/200) overnight at 4°C. Cells were washed with PBS/0.1%BSA before incubation with the secondary antibody (Alexa Fluor 494 goat anti-rabbit IgG, (red)) at 1/200 dilution for 1 h at RT. DAPI was used to stain the cell nuclei (blue). Cells were observed by epifluorescence microscopy (Axioplan2 imaging from Zeiss coupled to a cool SNAP HQ from Roper Scientific) using an objective Plan NeoFluar 40x /1.3.

Total RIPA extracts from melanoma cells were prepared. Samples were separated on 8% SDS-PAGE and transferred to nitrocellulose membranes (Amersham) using standard techniques. After 1 hr at room temperature (RT) in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 45 mmol/L NaCl, 0.1%Tween-20, and 4% non-fat milk), nitrocellulose membranes were incubated overnight at 4°C with rabbit anti-Patched antiserum (Ab39266 from Abcam 1/1000) and monoclonal mouse anti-βtubulin antibody (Sigma; 1/1000). After 3 washes, membranes were incubated 45 min with anti-mouse (1:5000) or anti-rabbit (1:3000) immunoglobulin coupled to horseradish peroxidase (Dako). Detection was carried out with an ECL kit (Millipore) on a Las3000 (Fuji).