Prolonged inhibition of LRRK2 induces macroautophagy and ULK1 phosphorylation on Ser758
Six hours (A) and eighteen hours (B) LRRK2in1 treatment to inhibit LRRK2 kinase. The gels shown are representative of three independent experiments. (C) Six, ten and eighteen hours of LRRK2in1 treatment, pSer758ULK1 was normalized against total ULK1. Quantification was done for three independent experiments; after normalization against the control in DMSO, data were pulled together in the dose–response curve (mean and standard error). Statistical analysis was performed with two-way ANOVA; *P<0.05 and ***P<0.001. (D) Eighteen hours treatment with LRRK2in1 in the presence and absence of bafilomycin-A1 to block the autophagy flux. The gel shown contains three replicates and is representative of three independent experiments as quantified in (E) mean and standard deviation; statistical analysis was performed by one-way Anova followed by Tukey’s post-hoc test. (F) Eighteen hours GSK2578215A treatment to inhibit LRRK2 kinase; the gel shown is representative of three independent experiments and it is quantified in (G/H) with mean and standard deviation. (G) Ser758-ULK1 and total ULK1 were quantified against their own β-actin loading control; then, Ser758-ULK1 was normalized against total ULK1. Statistical analysis was performed by unpaired, Student’s t-test. (H) Ser556-ULK1 and total ULK1 were quantified against their own β-actin loading control; then, Ser556-ULK1 was normalized against total ULK1. Statistical analysis was performed by unpaired, Student’s t-test. (I and L) Eighteen hours LRRK2in-1 treatment to inhibit LRRK2 kinase in primary rat astrocytes at 20 days in culture. The gels shown are representative of three independent experiments (14, 15 and 20 days in culture) quantified in (M) and (N). (M and N) Ser758-ULK1 and total ULK1 were quantified against their own β-actin loading control; then, Ser758-ULK1 was normalized against total ULK1. Variation over the control in DMSO (mean and standard deviation) is reported in the graphs Statistical analysis was performed by Student’s t-test; *P<0.05, **P<0.01 and ***P<0.001.