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. Author manuscript; available in PMC: 2023 Mar 16.
Published in final edited form as: Cell Rep. 2023 Jan 4;42(1):111969. doi: 10.1016/j.celrep.2022.111969

Figure 1. Delivery of endocytosed cargo to LEs/LYSs requires Rab7, Arl8a and Arl8b, HOPS, and BORC.

Figure 1.

(A) Scheme of pulse/chase labeling and purification of FF endosomes.

(B) Possible scenarios when analyzing WT versus KO FF endosomes. PNS, postnuclear supernatant; cyt, cytosol. See main text for details.

(C–J) FF endosomes purified from HeLa WT or KO cells and PNSs were analyzed for LAMP1 (C–F) or mature cathepsin D (mCathD; G–J) by immunoblotting.

(C) Representative LAMP1 immunoblots of PNSs and FF endosomes.

(D) Quantification of immunoblots as in (C). PNS and LYS samples were normalized to the PNS and LYS sample type with the strongest signal.

(E) Representative LAMP1 immunoblots of PNSs and FF endosomes from HeLa WT or Rab7 KO cells. Eletrophoretic mobility of LAMP1 was altered in PNSs and LYSs from Rab7 KO cells as reported previously.38

(G–J) Done as (C)–(F) but with mCathD antibody. A membrane (mem) fraction was used for whole-cell analysis (G and I).

(K–N) Endosomes purified from WT or KO HeLa cells after a 2 or 24 h chase of FF were analyzed for mCathD.

Data are means and SDs from 3 (D, H, L, and N) or 4 (F and J) independent experiments.