Dimer formation stabilizes monomeric subunits, maintaining a steady ER resource for assembly of γ-secretase complexes. (A) SDS-PAGE of subunit steady-state levels and degradation after CHX treatment in MEF WT, NCT-KO, APH1-tKO, PSEN-dKO, and PEN-2-KO. Representative blots after 0-h (steady state), 6-h, or 24-h CHX treatment (filled arrowheads: immature NCT; open arrowheads: FL PSEN1; open arrows: PSEN1-NTF). (B) Quantification for degradation normalized to 0 h and for steady-state levels to WT levels (0 h). Immature NCT degraded fastest and had the lowest steady-state levels in the absence of APH1 and vice versa. Likewise, PEN-2 degraded fastest and had the lowest steady-state levels in the absence of PSEN. FL PSEN1 degraded slowest and was most stable in the absence of PEN-2 (i.e., when NCT/APH1A/PSEN1 trimer formation was observed). Graphs show mean normalized protein levels ± SEM (n = 3) (C) Lateral (top) and bottom (bottom) views of the γ-secretase structure (Protein Data Bank accession no. 6LR4, prepared using UCSF Chimera; Pettersen et al., 2004) with NCT (green), APH1A (blue), PSEN1 (orange), and PEN-2 (pink). Asterisks indicate the connection between the PSEN1 C-terminus and APH1A; arrowheads indicate PSEN1–PEN-2 interaction; dotted lines indicate the space between dimers (Video 1).