Analysis of PLP endocytosis by cell surface biotinylation.

Surface proteins of GFP-MAL2/HOG cells were biotinylated at 4°C using a Sulfo-NHS ester of biotin. After biotinylation, cells were cultured in DM at 37°C for 150 min, fixed and treated for immunofluorescence analysis with anti-PLP monoclonal antibody and streptavidin-Alexa Fluor 555, to visualize biotinylated proteins. Monoclonal anti-PLP antibody was detected using an Alexa Fluor 647 secondary antibody. Images correspond to confocal slices of 0.8 µm, and the last one is the projection of confocal planes. Endocytosed streptavidin-positive proteins appear red. Arrows show endocytosed PLP (magenta) that colocalizes with endocytosed GFP-MAL2 (yellow). White vesicles correspond to endocytosed PLP colocalizing with endocytosed GFP-MAL2. Scale bar  = 5 µm.