Skip to main content
. 2023 Dec 13;9(50):eadj1205. doi: 10.1126/sciadv.adj1205

Fig. 1. Phospho-Rab and RILPL1 enrichment at VPS35[D620N] mutant lysosome.

Fig. 1.

(A) Workflow of the LysoTag IP methodology. (B and C) The indicated littermate-matched MEFs were transduced ± LysoTag (TMEM192-3xHA) and subjected to LysoTag IP. A sample of the homogenate was removed and designated WCL. Experiments were performed in six technical replicates and analyzed by DIA-MS, and the data are presented as Volcano plots (WCL Curtain link: https://curtain.proteo.info/#/0e673d58-d8f2-4368-996b-0869d5513d46 and lysosome Curtain link:https://curtain.proteo.info/#/0e673d58-d8f2-4368-996b-0869d5513d46 and table S4). The red dots represent the substantially differentiated proteins with fold change > 1.5 and P < 0.05, the green dots represent the LRRK2 pathway–related proteins, and the blue dots represent the lysosomal annotated proteins. (D) Violin plots of RILPL1 and VPS35 levels derived from experiment in (B) and (C). (E) Indicated MEFs transduced ± either the LysoTag (TMEM192-3xHA) or GolgiTag (TMEM115-3xHA) were subjected to organelle isolation as in (C) and (D). Two micrograms of both the immunoprecipitate and respective input (WCL) was subjected to immunoblot analysis using the LI-COR Odyssey CLx Western blot imaging and the indicated antibodies. Each lane indicates a sample derived from a different dish of cells. Quantitation of immunoblotting data (performed using ImageStudioLite software version 5.2.5, RRID:SCR_013715) is shown as mean ± SEM. (F) Indicated homozygous knock-in (KI) LysoTag (TMEM192-3xHA)–transduced MEFs were transfected with GFP-LRRK2. Colocalization of GFP-LRRK2 with TMEM192-3xHA was quantified and analyzed using Manders’ coefficient after automatic thresholding. Data from 36 technical replicates (12 cells from three biological replicates) are presented as mean ± SEM. Statistical analysis was performed using two-tailed unpaired Student’s t test (****P < 0.0001). Scale bars, 2 μm.