H4 cells were transfected with 2–10 μg LRRK2 shRNA or scramble Open Biosystems GIPZ shRNAmir (V3LHS-644167, Thermo Fisher Scientific) using Effectene (Qiagen) transfection reagent according to the manufacturer’s instructions. ShRNA vectors contain a puromycin resistance gene. Cells were treated with 2 μg/ml puromycin supplemented DMEM 48 h after transfection and kept under selection for expansion. Selection was removed 24 h before the experiment to avoid interference of the antibiotic with the treatment. (A) Eighteen hours of LRRK2in1 treatment. The gel shown contains three replicates; it is representative of three independent experiments and it is quantified in (B). (B) Ser758 ULK1and total ULK1 were first quantified against their own β-actin loading control; then, Ser758 ULK1 was normalized against total ULK1 and variation was calculated against the scrambled control in DMSO. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post-hoc test. (C) Staining for LRRK2 with the LRRK2 antibody MJFF#2, 3514-1/ab133474, Epitomics. The gel shown contains three replicates; it is representative of three independent experiments quantified in (B). (D) LRRK2 was normalized against β-actin loading control; mean and standard deviation are shown; statistical analysis was performed by unpaired, Student’s t-test. The knockdown led to a decrease in approximately 60% LRRK2 expression; *P<0.05 and **P<0.01.