Figure 1: ILEI is associated with mature γ-secretase complexes.

(a) SYPRO- and silver-stained SDS–PAGE gels of TAP-tagged PEN-2-associated proteins isolated from lysates of HEK-TAP-PEN-2 (P) and HEK-TAP-empty (E) cells. ILEI was identified in the band indicated by the star. (b) Co-immunoprecipitation of endogenous ILEI and γ-secretase components. Native HEK293 cell lysates were immunoprecipitated with normal IgG or an antibody against NCT, PS1, PEN-2 or APH-1. The blot was probed with anti-ILEI antibody (upper panel). As the FL ILEI bands were close to the IgG light-chain bands, nanocapsules incorporating IgG Fc-binding Z domains were used instead of the secondary antibody for detection (lower panel). The star indicates the IgG band. FL, full-length; SF, secreted form. (c) Reverse co-immunoprecipitation. HEK293 cell lysates were precipitated using normal rabbit IgG (IP-IgG) or anti-ILEI antibody (IP-ILEI). The blots were probed using the indicated antibodies. (d) Two-dimensional Blue Native (BN)/SDS–PAGE analysis of ILEI and γ-secretase components. The membrane fraction of native HEK293 cells was subjected to two-dimensional BN/SDS–PAGE before immunoblotting. (e) Chemical crosslinking of ILEI with γ-secretase components. HEK293 cells were crosslinked in situ with dithiobis succinimidyl propionate. The 1% Nonidet P-40-solubilized cell lysate was subjected to immunoprecipitation. A blot of the immunoprecipitates with antibodies against the indicated proteins or normal IgG was probed with anti-ILEI antibody. (f) The crosslinked cell lysates were immunoprecipitated using normal IgG or anti-ILEI antibody. The blots were probed using the indicated antibodies.