Gropro

Control or RPE1 cells stably expressing a small hairpin RNA targeting CMG2 mRNA were seeded on plates and incubated overnight in DMEM complemented with Gropro (Zen-Bio), to remove the collagen VI present in FBS. The next days, cells were incubated or not for 60 min with 100 nM bafilomycin (Sigma). Thirty minutes before the addition of purified collagen VI tetramer (kind gift from P. Bonaldo), 5% BSA was added to the cell culture medium to block aspecific binding. A measure of 1 μg ml⁻¹ of purified collagen VI tetramer was then added to cells. Cells were lysed in IP buffer (0.5% NP-40, 500 mM Tris-HCl pH 7.4, 20 mM EDTA, 10 mM NaF, 30 mM sodium pyrophosphate decahydrate, 2 mM benzamidin, 1 mM PMSF, 1 mM N-ethyl maleimide, cocktail of proteases inhibitors (Roche) and phosphatase inhibitors (Sigma)) 1, 3 and 6 h after collagen VI addition. Protein quantity was measured using BCA assay kit (Pierce) and equal amount of proteins boiled in Laemmli buffer under non-reducing conditions for 5 min before analysis by SDS–PAGE using 4–12% Bis-Tris gradient gels and western blotting with collagen VI antibody and rat monoclonal anti-CMG2 antibody. Actin was used as a loading control.