Figure EV3. Cross‐linking data demonstrate a short‐distance interaction between NCT 241 and APP_{C 99} K28.

• A
  Representative SDS–PAGE/Western blot analysis of CHAPSO‐solubilized membrane proteins extracted from Ncstn^−/− MEF cell lines stably expressing WT or mutant mNCT‐I241C subunits (shown in Fig 5). The presence of mature NCT, N‐terminal, and C‐terminal fragments of the endoproteolyzed PSEN1 as well as PEN‐2, compared to NCT knock‐out (KO) cells, indicates that WT and the I241C mutant reconstitute GSEC complexes. *Non‐specific band. Arrowheads indicate the position of molecular weight markers.
• B
  Representative SDS–PAGE/Western blot presenting NCT‐APP_C99/Aβ and APP_C99 expression (as detected by 82E1 anti‐Aβ antibody) in detergent‐extracted membranes prepared from MEF cell lines co‐expressing respective NCT‐I241C mutant GSEC complex with APP_C99‐K28C substrate. In the non‐reduced condition (‐ βME) bands corresponding to APP_C99 and/or Aβ dimers are appearing, as indicated with arrowheads. To estimate the amount of substrate cross‐linked to GSEC (i.e., spontaneous disulfide bond formation between APP_C99/Aβ and NCT), the NCT‐APP_C99/Aβ band was quantified and normalized to “total APP/Aβ expression” (defined as the sum of the bands indicated with arrowheads). Mean efficiency ± SD = 15.4 ± 5.7%, N = 3 independent experiments. Arrowheads indicate the position of molecular weight markers.
• C, D
  Analysis of the integrated density profiles on the full blots shown in Fig 5B (C) and 5E (D).

Source data are available online for this figure.