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. 2019 Oct 19;28(23):3982–3996. doi: 10.1093/hmg/ddz245

Figure 2.

Figure 2

LRRK2 is present in the nuclear cytoskeleton and interacts with lamin A/C in vitro and in vivo. HEK293 cells (A) and primary neurons (B) were fractionated into cytosolic and nuclear fractions. Nuclear fractions were separated into soluble and insoluble material, and the insoluble fraction further separated into chromatin-bound and nuclear cytoskeletal fractions. Levels of endogenous LRRK2 were determined with anti-LRRK2 (upper panels). The blots were re-probed for lamin A/C, histone 3 (A) or 2B (B). (C) Interaction of endogenous LRRK2 from rat brain lysate with GST-lamin A/C (aa 320–647). Binding of endogenous LRRK2 was determined using anti-LRRK2 (upper panel). The lower panel depicts lamin A/C fragments stained by Ponceau S. (D) Binding of Flag-LRRK2 from cell lysate with GST-lamin A/C fragments. The binding was determined with anti-Flag (upper panel). The lower panel depicts lamin A/C fragments stained by Ponceau S. The graph depicts the relative binding of Flag-LRRK2 to the various GST-lamin A/C fragments. Values represent the average ± S.E.M. of three independent experiments. Repeated-measures one-way ANOVA with Bonferroni post hoc test (P < 0.001). (E) Co-immunoprecipitation of Flag-LRRK2 with HA-lamin A/C from transfected HEK293 cells. Co-immunoprecipitation of LRRK2 was detected with anti-Flag (upper panel) while levels of lamin A/C immunoprecipitation were determined with anti-HA (middle panel). (F) HEK293 cells were transfected with Flag-LRRK2, in the presence of HA-lamin A/C or HA-lamin B1. Lamins were immunoprecipitated from cell lysates with anti-HA antibody (middle panel), and co-immunoprecipitation with Flag-LRRK2 was detected with anti-Flag (upper panel). The graph represents the relative co-immunoprecipitation of wild-type LRRK2 with lamin A/C in contrast to lamin B1. Values represent the average ± S.E.M. of three independent experiments. Unpaired two-tailed Student’s t test (P < 0.001). (G) Lamin A/C was immunoprecipitated from rat brain using the anti-lamin A/C antibody (lower panel) and co-immunoprecipitation with endogenous LRRK2 determined with the anti-LRRK2 antibody (upper panel).