(A) AE-MS of HA-Rab8A and (B) endogenous Rab8A using extracts of GFP-LRRK2-R1441G expressing Flp-In T-Rex HEK293 cells. Expression of the kinase was induced for 48 hr by doxycycline (1 µg/ml) and LRRK2 inhibited using HG-10-102-01 (3 µM, 3 hr). LRRK2-regulated, known and unknown Rab8A-binding proteins in both AE-MS experiments are highlighted in red. (C) Label-free (LFQ [Cox et al., 2014]), MS-quantified RILPL1 and RILPL2 levels after immunoprecipitation of Rab8A from mock- or MLi-2 (200 nM, 2 hr) treated LRRK2-R1441C MEFs. (D) Pulldown of GFP-tagged RILP, RILPL1 or RILPL2, transiently expressed with HA-Rab8A and the indicated LRRK2 variants (KD= Y1699C/D2017A) in HEK293 cells. Western blot after Phos-tag SDS-PAGE was used to detect interacting proteins using the indicated antibodies. (MLi-2 = 150 nM, 2 hr). (E) Sequence alignment of the RILP homology (RH) domains of RILP, RILPL1 and RILPL2 showing five conserved basic residues, which are highlighted. (F) Same as (D) but using different RILPL1 and RILPL2 mutants. For phos-tag blots, filled circles indicate non phosphorylated proteins and open circles phosphorylated proteins. (G) AE-MS of GFP-RILPL1 (wt or R291E) and (H) GFP-RILPL2 (wt or R130E), expressed with LRRK2-Y1699C in HEK293 cells, and treated or not with MLi-2 (for wt, 200 nM, 2 hr). The student’s two sample test statistic (Log2) of the indicated comparisons was used for plotting.