(A, upper panel) HEK293 Flp-In T-REx cells stably expressing wild type (WT) GFP-LRRK1 were treated with 1 µg/ml doxycycline for 24 h to induce expression of GFP-LRRK1. Cells were then serum-starved for 16 h, incubated ± the indicated concentration of LXS-196, GÖ6983 or CRT0066051. Thirty minutes prior to lysis, cells were stimulated ± 160 nM phorbol 12-myristate 13-acetate (PMA) for 30 min and cell lysed. Upper panel- GFP-LRRK1 immunoprecipitated using an anti-GFP nanobody and LRRK1 kinase activity towards recombinant Rab7A assessed in a 30 min assay. Reactions were terminated by addition of SDS-sample buffer and levels of pSer72-Rab7A, Rab7A and LRRK1 were assessed by quantitative immunoblot analysis using the LI-COR Odyssey CLx Western Blot imaging system with the indicated antibodies. Combined immunoblotting data from two independent biological replicates (each performed in duplicate) are shown. (A, lower panel) Quantified immunoblotting data of LRRK1 kinase assays are presented as ratios of pRab7ASer72/total Rab7A (mean ± SEM) vs concentration of LXS-196 relative to levels observed with no inhibitor added (100%). IC₅₀ values were calculated with GraphPad Prism (version 9.1.0) using non-linear regression analysis. Note that the blank value of basal LRRK1 activity observed in unstimulated cells with no inhibitor was subtracted from stimulated values for the IC50 analysis (B) The whole cell extracts (10 µg) that were prepared in (A) were subjected to quantitative immunoblot analysis using the LI-COR Odyssey CLx Western Blot imaging system with the indicated antibodies. (C) As in (A) except wild type and homozygous LRRK1 knock-out primary Mouse Embryonic Fibroblasts (MEFs) were serum-starved for 16 h and stimulated ± 160 nM PMA for 30 min and cells lysed. Whole cell extracts (10 µg) were prepared and subject to quantitative immunoblot analysis. (D) As in (C) except, endogenous LRRK1 was immunoprecipitated from whole cell extracts using a polyclonal total LRRK1 antibody and LRRK1 kinase activity towards recombinant Rab7A assessed in a 30 min assay. Phosphorylation off Rab7A was assessed by immunoblot analysis.