Prior to western blot analysis, Dhh, Ptch1 ou Smo were immunoprecipitated with mouse anti-Dhh antibodies (Santa Cruz Biotechnology, Cat# sc-271168), anti myc-tag antibodies (Millipore, Cat# 05-724) or mouse anti-Smo antibodies (Santa Cruz Biotechnology, Cat# sc-166685). Expression of Cdon, Gas1, Dhh, Ptch1 and Smo were evaluated by SDS PAGE using goat anti-mouse Cdon antibodies (R&D systems, Cat#AF2429), goat anti-human Gas1 antibodies (R&D systems, Cat# AF2636), mouse anti-Dhh antibodies (Santa Cruz Biotechnology, Inc, Cat#sc-271168), rabbit anti-Ptch1 antibodies (Abcam, Cat#ab53715) and mouse anti-Smo antibodies (Santa Cruz Biotechnology, Cat# sc-166685) respectively. Expression of human ICAM-1 and VCAM-1 expression were evaluated by SDS PAGE using mouse antihuman I-CAM1 antibodies (Santa Cruz Biotechnology, Cat#sc-8439) and rabbit anti-VCAM-1 (Abcam, Cat# ab134047) respectively. Protein loading quantity was controlled using a monoclonal anti-α-tubulin antibody (Sigma). Secondary antibodies were from Invitrogen, Cat#A-21039, A-21084, A-21036). The signal was then revealed by using an Odyssey Infrared imager (LI-COR).
In vitro permeability assay 100 000 cells were seeded in Transwell® inserts. The day after, 0.5 mg/mL 70 kDa FITC-Dextran (Sigma) was added to the upper chamber. FITC fluorescence in the lower chamber was measured 20 minutes later.
In vitro permeability assay 100 000 cells were seeded in Transwell® inserts. The day after, 0.5 mg/mL 70 kDa FITC-Dextran (Sigma) was added to the upper chamber. FITC fluorescence in the lower chamber was measured 20 minutes later.