Figure 3.

LRRK2 induction by TLR4 stimulation. A, Five micrograms of LPS or no LPS control was bilaterally injected into the SNpc of FLAG-LRRK2 mice (Jackson Laboratory strain 012466), the SNpc was dissected after 24 h, and FLAG-LRRK2 was immunoprecipitated and treated with ATP. Eluted protein was analyzed by Western blot with either a total LRRK2 antibody- or autophosphorylation-specific pT1503 antibody. B, Quantification of pT1503-autophosphorylated LRRK2 normalized to total LRRK2 from four LPS-injected mice and four injection control mice. C, Specificity of the pT1503 antibody is demonstrated by LRRK2 recombinant protein derived from transiently transfected HEK-293FT cells. D, Primary cultures derived from P2 rats were analyzed by Western blot for cell type-specific markers and LRRK2 expression. Lysates were normalized to actin, and ∼20 μg of protein was loaded per lane. E, F, Primary microglia treated with various concentrations of LPS for 12 h and LRRK2 expression evaluated by Western blot (E), with quantification normalized to actin for three independent experiments (F). G, mRNA levels of LRRK2 were determined by relative quantification (ΔΔcT) normalized to TBP. H, Representative immunofluorescence of LRRK2 staining in primary microglia cultures treated with LPS or control (-LPS) for 12 h. I, Human LRRK2 expression characterized by Western blot in human primary microglia cultures compared with human macrophage/monocyte THP-1 cells. J, THP-1 cells treated with 100 ng of LPS for the indicated time and lysates analyzed by Western blot (J), with quantification of LRRK2 levels from three independent experiments (K). *p < 0.01, two-tailed unpaired t test (B); *p<0.01, one-way ANOVA with Tukey–Kramer test (B, F, K), with respect to initial LRRK2 expression. Error bars indicate SEM.