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. 2019 Jan 15;14(1):e0210248. doi: 10.1371/journal.pone.0210248

Fig 1. Mn increases LRRK2 kinase activity and expression in RAW 264.7 and HMC3 cells.

Fig 1

(A) Two-hundred (200) μg of protein were collected from cell lysates, followed by western blot analysis to determine the presence of LRRK2 in LRRK2 WT, KO RAW 264.7 and HMC3 cells. β-actin was used as a loading control. (B,C) LRRK2 WT or HMC3 cells were treated with Mn (250 μM) for the designated times, followed by protein extraction and western blot analysis as described in the Methods section. Protein levels of LRRK2 and phosphorylated LRRK2 (S1292) in LRRK2 WT RAW 264.7 (B) and HMC3 (C) cells were quantified. (D,E) Effect of Mn on LRRK2 mRNA levels in LRRK2 WT RAW 264.7 (D) and HMC3 (E) cells were assessed as described in the Methods section. GAPDH was used as a loading control. (F) After pre-treatment with LRRK2 inhibitors GSK (1 μM) and MLi-2 (50 nM) for 90 min, RAW 264.7 cells were exposed to Mn (250 μM) for 20 min, followed by western blot analysis to detect phosphorylation of LRRK2 (S1292). ###, p < 0.001; *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to the control (one-way ANOVA followed by Tukey’s post hoc test; n = 3). The data shown are representative of 3 independent experiments.