N-glycosylation of PRG3 at asparagine 163 is required for membrane insertion and filopodia formation. (A) Western blot of mouse brain lysates probed with anti-PRG 53 before and after incubation with N-glycosidase F. When treated with N-glycosidase F a band is detected at a lower molecular weight compared to untreated lysate, pointing to an N-glycosylation of PRG3. (B) Western blot of HEK293 cells after transfection with two different PRG3 constructs (3xFLAG-rPRG3 and 3xFLAG-rPRG3 N163Q). Native lysates (lanes 1 and 3) or lysates treated with N-glycosidase F (lanes 2 and 4) were probed with an anti-FLAG antibody. Mutation of asparagine 163 to glutamine (lanes 1 and 2) almost completely abolished N-glycosylation of rPRG3 whereas native protein was still N-glycosylated (lanes 3 and 4). (C) Confocal images of representative HEK293 cells transfected with either 3xFLAG-rPRG3 (left) or 3xFLAG-rPRG3 N163Q (middle) in comparison to control-transfected cell (empty vector) (right). HEK293 cells overexpressing the mutated rPRG3 construct show no increase of filopodia formation, which are prominently visible when HEK293 cells express the native rPRG3 variant. Cells were stained with a primary anti-FLAG and a secondary alexa488 antibody. Scale bar: 10 μm. White rectangles indicate the areas chosen for higher magnification displayed in the left lower left corner. Cells were visualized with fluorescence, laser transmission and combined in an overlay (shown). HEK293 cells expressing 3xFLAG-rPRG3 exhibited filopodia and rPRG3 was inserted in the membrane (left). In contrast, 3xFLAG-rPRG3 N163Q and empty vector control showed no localization in the plasma membrane and filopodia were not visible. (D) Co-localization images show wild-type PRG3 co-localized with the surface marker Na+/K+-ATPase, whereas mutated PRG3 shows no co-localization. Scale bar: 5 μm.