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Following gene ontology analysis of protoarray hits, proteins categorized as membrane bound organelles were identified. Potential hits in addition to CSNK1A1, ARHGEF7 and NTC were used to order a custom pooled ON-TARGETplus siRNA library (Dharmacon). HEK293FT cells were plated in 96 well plates and reverse transfected with siRNA at a final concentration of 50nM. Twenty-four hours later, cells were transfected with 3xFlag WT LRRK2 together with either 2xmyc WT RAB29 or 2xmyc Q67L RAB29 using Lipofectamine 2000. Thirty hours following plasmid transfection, cells were fixed and stained for M2-Flag, Myc (clone 9E1) and TGN46 before labeling with fluorescent secondary antibodies and Hoechst 33342 (Thermo Scientific). Cells were imaged at 20x objective using a Cellomics VTI Arrayscanner and the percentage of cells with Flag WT LRRK2 and TGN46 positive spots were recorded. For each siRNA, 6 wells containing a minimum of 1000 cells each were analyzed. Samples were compared to NTC siRNA control and ranked.

HEK 293FT cells were seeded at 1×10⁴ cells per well in a 96-well Matrigel coated plate. Cells were transfected by reverse transfection with pooled ON-TARGETplus siRNA against ARHGEF7, CSNK1A1or non-targeting control (Thermo) at a final concentration of 25 nM. Transfection was carried out according to the manufacturer’s protocol, using DharmaFECT 1 transfection reagent (Thermo). 24 hrs after siRNA transfection, cells were further transfected with 3xFLAG -LRRK2 and 2xmyc-GUS or 2xmyc-Rab7L1 WT or 2xmyc-Rab7L1 Q67L mutant plasmids using Lipofectamine-2000 (Life Technologies) reagent. 48 hrs after siRNA transfection and 24 hrs after plasmids transfection cells were fixed with 4% paraformaldehyde containing 1 µg/ml Hoechst and stained for endogenous TGN46 (AbD Serotec), FLAG (Sigma) and myc (Roche) antibodies. Plates were imaged at 20x objective on high throughput Cellomics VTI arrayScanner and analyzed using Spot Detector bioapplication for % of cells with LRRK2 and TGN46 positive spots from total number of LRRK2 transfected cells. Three independent experiments were performed with 6 wells per sample with the minimum of 1000 cells/well. siRNAs samples for Rab7L1, ARHGEF7, or CK1α were compared to NTC within families (GUS, Rab7L1 WT, or Rab7L1 Q67L) by two way ANOVA, Tukey’s multiple comparison test.