PS dependent γ-secretase cleavage of SorCS1bV5 in lipid rafts. a) Mouse embryonic fibroblasts that are PS-/- were transiently transfected with SorCS1bV5 in the same fashion as figure 3a. Cells transiently transfected with sorCS1bV5 produce a ~130 kDa holoprotein labeled by anti-V5. In the absence of PS1 or the presence of a dominant negative PS1 mutant (D385E) the levels 13 kDa SorCS1bV5 CTF are markedly increased. However, cotransfection of PS1 wt or PS1 familial Alzheimer's disease linked mutant (M139V) and SorCS1bV5 lead to a reduction in the levels of 13 kDa anti-V5 positive SorCS1bV5 CTF. Anti-β-actin was included as a loading control and anti-PS1ct antibody demonstrated that PS1 wt and M139V underwent endoproteolysis whereas D385E did not. b) HEK cells stably overexpressing sorCS1bV5 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 faint SorCS1bV5 CTF was detected in fractions 4 and 5 of the 37°C incubated sample. Anti-V5 antibody detected the SorV5 holo protein at 130 kDa, a CTF at 13 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. c) HEK cells stably overexpressing sorCS1bV5 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 SorCS1bV5 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 not detected as a result of incubation at 37°C but the disappearance of the 13 kDa SorCS1bV5 CTF was blocked by γ-secretase inhibitors. d) Samples were prepared as in b). Disappearance of the SorCS1bV5 CTF was inhibited in a dose dependant fashion by a γ-secretase inhibitor, LY411,575. Anti-PS1NT demonstrates the loading consistency.