Fig 2. Comparison of kinase activity of LRRK2 variants and pathogenic mutants.

a) Quantification of phosphorylated LRRKtide was performed by ELISA using phospho-specific LRRKtide antibodies. Subsequently, the captured Flag-LRRK2 was quantified by an ELISA using a N-terminal targeted anti-LRRK2 antibody. The amount of phosphorylation of LRRKtide was directly normalized to the amount of LRRK2 present within the same well. * p<0.05 in comparison to WT; *** p<0.001 in comparison to WT. b) To determine the in-well auto-phosphorylation activity of LRRK2 at the Ser1292 residue, kinase reactions were carried in Flag-captured ELISA plates in kinase reaction buffer in the presence or absence of 100 μM ATP, without additional substrate added. The data were normalized to parallel wells processed for total LRRK2 by ELISA. In the absence of ATP, the signal represents the basal levels of Ser1292 auto-phosphorylation; whereas in the presence of ATP, the induction of auto-phosphorylation activity at this residue is observed. The data points in (a) and (b) represent the mean of at least three independent experiments, with duplicate wells per sample within each individual experiment. c) representative Western immunoblots showing the specificity of the pS1292 antibody, the absent signal in extracts from cells expressing S1292A-LRRK2 (left blot). In the right panel, the relative degree of cellular phosphorylation levels of S1292 as determined by Western immunoblot using the same antibody, and the corresponding level of Flag-LRRK2 expression using anti-Flag antibodies. When normalized across at least 3 independent experiments, an induction in basal S1292 phosphorylation is only significantly elevated in cells expressing the pathogenic mutants I2020T or G2019S-LRRK2 (d). *** p<0.001 in comparison to the absence of ATP; ### p<0.001 in comparison to WT in presence of ATP; +++ p<0.001 in comparison to WT in absence of ATP.