γ-Secretase complexes enrich from ERGIC to endosomal fractions and PM (see also Fig. S1 ). (A) BN-PAGE of γ-secretase complexes in PNS compared with enriched compartments (ER, ERGIC, and early to late endosomes/lysosomes [endo]) from MEF WT and PSEN-dKO (see Fig. S1). Membranes were extracted in 0.5% DDM to resolve complexes (Fraering et al., 2004a; Spasic et al., 2007), and equal protein amounts were loaded. Western blot for NCT, APH1A_L, PSEN1-CTF, and PEN-2 showed a gradual increase in the ratio of the 440 kD band (full complex; filled squares) versus ∼220 kD NCT/APH1A subcomplexes (asterisks), particularly between the ER and the ERGIC. Mature complexes were predominant in endosomal fractions. In PSEN-dKO, only NCT/APH1A subcomplexes were found in the ER and were most strongly enriched in the ERGIC. APH1B-containing complexes dominated in endosomal fractions. (B) BN-PAGE of DDM extracts of PNS and isolated PMs (Tharkeshwar et al., 2017). Only ∼440 kD full complexes (filled squares) were found positive for all subunits, including APH1A, being highly abundant at the cell surface. Open circles indicate the NCT/APH1A/PSEN1-CTF subcomplex. Representative Western blots are shown.