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. 2018 Feb 28;11:64. doi: 10.3389/fnmol.2018.00064

Figure 3.

Figure 3

E193K variant affects LRRK2 phosphorylation at Ser935. (A) Protein lysate prepared from primary fibroblast obtained from healthy individuals or E193K carrier was analyzed by western-blotting to appreciate total LRRK2 level and phosphorylation at Ser-935. (B,C) The graphs report LRRK2 total level, expressed as optical density and normalized vs. β-tubulin amount (B) and P-Ser935 level, expressed as optical density and normalized vs. total LRRK2 amount (C). Data are shown as mean ± SE, n = 8; ***p < 0.001 vs. WT. (D) N2a cells expressing FLAG-14-3-3ε together with myc-LRRK2 WT or myc-LRRK2 E193K variant were solubilized and processed for FLAG immunopurification. We evaluated the extent of LRRK2 binding to 14-3-3ε by measuring the amount of myc LRRK2 co-precipitating with FLAG 14-3-3ε (E) The graph reports the amount of myc-LRRK2 variant recovered in FLAG immunoprecipitates. Data were normalized to the amount of 14-3-3ε immunoprecipitated and expressed as mean ± SE, n = 4; **p < 0.01versus WT. (F) N2a cells expressing GFP together with RFP-LRRK2 WT or RFP-LRRK2 E193K variant were processed for imaging purposes. Scale bar = 10 μm.