Fig 5. Effect of expression of the full-length PLXNA4 isoforms (TS1, TS2 and TS3) on APP processing.

HEK293 cells stably overexpressing APP were transiently transfected with empty vector control (EV) or with the full-length (TS1) or one of the shorter isoforms (TS2 and TS3) of PLXNA4-Myc. 48 hrs after transfection, the conditioned medium (medium) and the cell lysates (lysate) were collected and analyzed by SDS-PAGE and western blotting using mAb 6E10 for total APP and APPsα, Myc mAb for all 3 isoforms of PLXNA4, and tubulin as control (A). The endogenous myc can be seen in all lanes at ~55 kDa but more clearly in lanes 7–9 where only empty vector was transfected but not PLXNA4-myc. Also note that TS1 migrates at 212kDa, TS2 migrates at 58kDa and TS3 migrates at 55kDa. (B) Densitometric analysis of the expression of APPsα normalized to total APP. Error bars indicate standard deviation. (C) ELISA analysis of Aβ₄₀ and Aβ₄₂ released to the medium. ELISAs were carried out using the human Aβ₄₀ and Aβ₄₂ ELISA kits (Invitrogen) in accordance with manufacturer's protocol. Error bars indicate standard deviation. Representative results of three independent experiments are shown in each panel.