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. 2020 Nov 3;11:5559. doi: 10.1038/s41467-020-19032-0

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — a Immune-precipitation of HA-tagged NPC1 and detection of NPC1-interacting proteins using mass spectrometry. Most abundant interaction partners detected with >2 peptides are indicated (full dataset available in Supplementary Data 2 and via ProteomeXchange with identifier PXD021444). b Immune precipitations of HA-tagged wild-type, dominant-negative (T22N) or constitutively active Rab7 (Q67L) show an activation-dependent interaction between Rab7 and endogenous NPC1 (two independent experimental replicates). c, d Reciprocal immune-precipitation of endogenous NPC1 (c) or Rab7 (d) shows a strong interaction between both proteins. e The Rab7-NPC1 interaction is lost in MCC-deficient cells that lack Rab7 activation. Wild-type, C18orf8-, Ccz1- and Mon1A/B-deficient cells were stably transduced with the inactive NPC1-P692S-HA. HA-tagged NPC1 was immune precipitated and immune blotted for endogenous Rab7. The inactive NPC1-P692S was used to prevent altering lysosomal cholesterol content by NPC1 overexpression. (two independent experimental replicates) f, g The Rab7-NPC1 interaction is independent of NPC1 activity or lysosomal cholesterol levels. f NPC1-deficient cells were complemented with HA-tagged wild-type or inactive P692S-mutant NPC1 and NPC1-HA immune-precipitations were analysed by immune blotting for endogenous Rab7. g Wild-type NPC1-HA complemented cells were treated with LPDS to decrease, or U18666A to increase lysosomal cholesterol levels and the NPC1-Rab7 interaction was probed using immune precipitation (two independent experimental replicates).