Cell line construction and maintenance for Lyso-IP and Endo-IP analysis of amyloid precursor protein processing, version 2 v2

• DOI: 10.17504/protocols.io.4r3l24kxxg1y/v2
• Publication Date: 2022-03-31T20:45:01Z
• Authors: Hankum Park; Frances V Hundley; Sharan Sharan Swarup; Harper JW

Lyso-IP is a method that allows for the isolation of lysosomes proteomics and metabolomics (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36). We have developed an analogous approach purification early/sorting endosomes (Endo-IP). In addition, we found endolysosomal via Endo-IP can be coupled to quantitative workflow obtain snapshots Amyloid Precursor Protein (APP) processing its Aβ products (Park et al. 2022). Here, describe methods cell line construction maintenance 293 cells with TMEM192-3xHA 3xFLAG-EEA1, which are used lysosome endosome purification, respectively. patient mutations APP promotes processing. Cells endogenously-tagged TMEM192 stably expressing FLAG-EEA1 referred as 293EL cells, Lyso-IP. These were also prepared in form has deletion gene (293EL APP-/-) same reconstituted lentivirus mutant (APPSw;T700N) functional analysis