Sucrose gradient fractionation of SorV5 overexpressing cells demonstrates enrichment of the 16 kDa CTF in buoyant "lipid raft" fractions and generation of a smaller SorV5 CTF. a) HEK cells stably overexpressing sorV5 were sucrose gradient fractionated and 1 ml fractions were collected from the top. Each fraction was analyzed by SDS PAGE on 12% XT Bis-Tris SDS PAGE (MES Buffer) and Western blotting. "4°C" were samples that were maintained at 4°C until SDS PAGE loading buffer was added. "37°C" indicates samples that were incubated at 37°C for 2 hours and then prepared as western samples. A smaller SorV5 CTF was detected in fractions 4 and 5 of the 37°C incubated sample. Anti-V5 antibody detected the SorV5 holo protein at 95 kDa, a CTF at 16 kDa and a smaller CTF in the "37°C" lanes 4 and 5. Anti-GS27 was used as a lipid raft marker (fractions 4–5), Anti-EEA1 is an early endosomal marker that was not found in lipid raft. PS1 CTF and NTF were enriched in raft fractions 4 and 5. b) HEK cells stably overexpressing sorV5 were pretreated with a γ-secretase inhibitor (IL-aldehyde) for 30 hours and sucrose gradient fractions 4, 5 were combined and analyzed for generation of the smaller SorV5 CTF. Samples were equally divided and incubated at 37°C (4°C for control) in the absence or presence of γ-secretase inhibitors, LY411575, and Compound E. A smaller SorV5 CTF band was detected as a result of incubation at 37°C but was blocked by γ-secretase inhibitors. c) Samples were prepared as in b). Generation of the smaller SorV5 CTF was inhibited in a dose dependant fashion by a γ-secretase inhibitor, LY411,575. Anti-PS1NT demonstrates the loading consistency.