Topology of the amino and carboxyl terminals of Fpn. HEK293T cells were plated on glass coverslips and transfected with pFpn-GFP or pFLAG-Zebrafish-Fpn (FLAG-ZFpn). Eighteen to 24 hours after transfection, cells were fixed using 3.7% formaldehyde in PBS and permeabilized (perm) (+) or not (-) using 0.1% saponin in PBS/BSA for 20 minutes. Cells were incubated with primary antibodies (mouse anti-Lamp1, rabbit anti-GFP, or mouse anti-FLAG) followed by Alexa 594–conjugated goat anti–mouse IgG or goat anti–rabbit IgG. (A) Shows the Fpn-GFP signal and the immunodetection of GFP only upon permeabilization with Lamp1 as a positive control for permeabilization. (B) Shows that the carboxyl and amino terminal FLAG epitopes on Fpn are detected only upon permeabilization.