Additional information regarding the LYSO-IP unbiased proteomics screening. (A and B) HEK293T cells stably expressing GFP-LRRK2 and TMEM192-3xHA were seeded for 24 h and treated with DMSO or LLOME for 2 h. Cells were then fixed and stained using an anti HA antibody. (C and D) HEK293T cells stably expressing GFP-LRRK2 and TMEM192-3xHA were seeded for 24 h and treated with DMSO, LLOME (2 h), and LLOME+MLi2. Cells were then fixed and stained for HA and endogenous RAB10 (C) or endogenous JIP4 (D). (E, F, and H) HEK293T cells stably expressing GFP-LRRK2 and TMEM192-3xHA were seeded for 24 h and treated with DMSO, LLOME (2 h), and LLOME+MLi2. Cells were then subjected to LYSO-IP to immuno-isolate lysosomes. Isolated lysosomes were lysed, and immunoblots show the amount of different cellular compartments in the lysosomal fraction, including EE, ER, cytosol, Golgi, RE, and mitochondria (E), as well as different luminal and membranous lysosomal proteins (F). (G) Marker quantification confirming enrichment of lysosomal proteins in the lysosomal fraction. (H) Immunoblot from LYSO-IP of players involved in LYTL, including pT73-RAB10, total RAB10, LRRK2, and JIP4. (I) Volcano plot showing the proteins with enhanced recruitment to lysosomes (right side) and the proteins decreased on lysosomes (left side), when cells are treated with LLOME. Data are from four independent experiments. (J) Histogram shows protein levels measured by mass spectrometry of individual proteins on lysosomes under DMSO, LLOME, and LLOME+MLi2. Data are from four independent experiments. (K) Immunoblot from LYSO-IP experiment showing that RILPL1 recruitment to damaged lysosomes is LRRK2 kinase activity dependent. Source data are available for this figure: SourceData FS1.