Figure 7.
LRRK2 contributes to autophagic turnover of aggregated protein in microglial cells. (A) Representative images of BV2 Scr and LRRK2 shRNA cells demonstrating the expression pattern of GFP-tagged proteins. GFP-Q23, a native derivate of Q74, was used as a control for protein expression. Western blot of soluble GFP-Q23 and GFP-Q74 demonstrates equality of transfection efficiency between Scr and LRRK2 shRNA cell lines. (B) BV2 Scr and LRRK2 KD cells were transiently transfected with GFP-Q74 construct for 24 h, with wells getting either vehicle or rapamycin (1 μm) for the last 12 h. Cells were sequentially extracted with 1% Triton X-100, to obtain soluble proteins, followed by 2% SDS, to isolate insoluble proteins. Insoluble protein solutions were protein normalized and separated by SDS–PAGE electrophoresis prior to western blotting using a GFP-antibody. Soluble proteins were also separated and immunoblotted for LRRK2 and actin as a loading control. Q74 clearance was quantified by densitometry and analyzed by one-way ANOVA (*P < 0.05). (C) An example of high molecular weight aggregates (‘gel-excluded GFP-Q74’) following 1% Triton X-100 extraction (‘TrX sol’) or 2% SDS extraction (‘insol’). BV2 Scr shRNA and LRRK2 shRNA cells were transiently transfected with Q74 for 24 h and then treated with vehicle or rapamycin (1 μm, last 12 h of 24 h transfection) prior to immunoblotting. (D) BV2 and MEF cell lines were transfected with Q74 constructs for 24 h prior to total protein extraction with 2% SDS. Cell lysates normalized for protein were spotted onto PVDF membrane prior to immunoblotting for insoluble GFP-Q74. Quantification is taken from three separate experiments, performed in triplicate (n = 9, Student's t-test, ***P < 0.0001). (E) BV2 cells were transfected with GFP-Q74 protein for 12 h prior to treatment with vehicle, GSK257A (1 μm) or LRRK2-IN-1 (1 μm). Following treatment, cells were sequentially lysed as previously. Quantification is from three separate experiments, each performed in triplicate (n = 9, one-way ANOVA, Dunnett's post hoc; *P < 0.05; **P < 0.01).