Solubilized membrane or surface protein fractions were denatured in 50 mM phosphate buffer, supplemented with 1% SDS and 0.5 M β-mercaptoethanol and afterwards incubated with endoglycosidase H or PNGase F (both Roche) in the presence of 0.5% NP-40 and protease inhibitors overnight at 37°C. Protein samples were finally resolved by SDS-PAGE and Western blotting.
