Figure 6.

CHIP knockdown increases protein expression levels of WT, G2385R, and ΔC LRRK2 variants.

HEK293FT cells were transiently transfected with smart pool NTC or CHIP siRNA (Thermo Scientific) 24 h before transfection with an equal amount of 3xFLAG-HD-LRRK2 plasmid DNA. After 48 h from transfection with LRRK2 constructs, cells were treated for 3 h with DMSO or 1 μM GA prior to lysis. (A) Co-immunoprecipitation of Hsp90, Hsc70, and CHIP with 3xFLAG-HD-LRRK2 constructs. (B) Adjusted steady-state levels of WT, G2385R, and ΔC LRRK2 protein expression in the presence of NTC (−) or CHIP (+) siRNA in input. (C–E) CHIP, Hsc70, and Hsp90-binding ability of different LRRK2 mutants normalized to the amount of immunoprecipitated LRRK2 (n = 6).