Journal: Cellular and Molecular Life Sciences
Article Title: APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms
Figure Lengend Snippet: APP wt homodimers are formed in the ER but also occur at the cell surface. a N2a cells were transiently co-transfected with NT HA APP695 CT Split GFP 1–10 and NT HA APP695 CT Split GFP 11. The cells were fixed with paraformaldehyde and immunostained with ER marker Grp78, followed by incubation with AlexaFlour-594 conjugated secondary antibody. The green fluorescence represents the APP dimer, induced by bimolecular fluorescence complementation (BiFC). Co-staining with Grp78 revealed colocalization of the APP dimer ( inset ) with the ER. b Single transfections of N2a cells with either NT HA APP695 CT Split GFP 1–10 or NT HA APP695 CT Split GFP 11. APP expression was verified with HA antibody, confirming a non-perturbed distribution ( right panel , red ). The green channel does not show any specific fluorescence. Scale bar is 10 μm. c N2a cells were transiently transfected with NT HA APP695 CT Split GFP 1–10, NT HA APP695 CT Split GFP 11 or GFP only. Cell lysates were analyzed via Western blotting for APP full length ( top part ), APP CTFs ( middle part ) and GFP, or GFP-containing CTFs ( bottom part ). Note that GFP antibody does not recognize GFP 11 alone. d CHO-K1 cells stably overexpressing APP 695 wt were pretreated for 1 h at 37°C in the presence of 10 μg/ml brefeldin A (BFA), or DMSO (vehicle) for 1 h to block protein secretion from the ER. Subsequently, cells were either directly lysed (BFA), or medium containing BFA was removed, and cells were incubated for additional 30 min at 37°C with 10 μM chlorpromazine (BFA/CPZ), to allow transport to the cell surface, but inhibit further endocytosis. Lysates were analyzed either under non-reducing (+β mercaptoethanol; −95°C), or reducing conditions (+β-mercaptoethanol; +95°C). Note that cells pretreated with BFA, followed by chlorpromazine exposure (BFA/CPZ), display two upper SDS-resistant bands migrating with a slight size shift, indicating different glycosylation patterns (dimer i/m). Note that strong denaturing conditions (+95°C) result in the disappearance of the upper migrating bands, suggesting disulfide bond formation. The separating line indicates different exposure times to visualize dimer formation in treated cells, whereas APP ER overexpressing cells served as size reference for dimers. e Cells were treated as described under d , and subjected to cell surface biotinylation to confirm drug treatments. APP ER is completely absent from the surface, whereas only minor quantities of mature APP reach the surface upon BFA treatment. Removal of BFA, and subsequent incubation with chlorpromazine (BFA/CPZ), strongly increases surface exposed mature APP, compared to vehicle control. Note that no dimers are recovered in biotinylation, because NeutrAvidin beads were boiled in BME containing sample buffer, to release biotin conjugated proteins
Article Snippet: Alternatively, samples were incubated with sample buffer without β-mercaptoethanol and heat denatured for 10 min. Proteins were electrophoresed on 4–12% NuPage (Novex®, Invitrogen) gradient gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA).
Techniques: Transfection, Marker, Incubation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Staining, Expressing, Western Blot, Stable Transfection, Blocking Assay