Figure 4

Loss of function of DJ-1 increased the aggregation of MAP1b-LC. A, DJ-1 shRNA vector or scramble shRNA vector was transfected into SH-SY5Y cells and selected with 300 μg hygromycin. The stable clone was picked and amplified. Western blot result showed efficient down-regulation of DJ-1. B, Flag-MAP1b-LC was transfected into DJ-1 shRNA or scramble SH-SY5Y cells for 48 hrs. Cells were lysed and separated into Triton X-100 soluble or insoluble components. The result showed increased insoluble MAP1b-LC in the DJ-1 KD cells. C, Flag-MAP1b-LC was transfected into DJ-1 shRNA SH-SY5Y or scramble shRNA cells for 48 hrs. Cells were immuno-stained with rabbit anti-Flag antibody. The results showed the enhancement of MAP1b-LC formed aggregates in DJ-1 KD cells. D, The total lysates of DJ-1 KD SH-SY5Y cells or scramble controls were separated into Triton X-100 soluble or insoluble fractions. Increased insoluble MAP1b-LC in DJ-1 KD cells was observed by Western blot. E, Endogenous MAP1b-LC was examined by immunofluorescence assay in DJ-1 KD SH-SY5Y cells or scramble controls. The MAP1b-LC formed aggregates increased in DJ-1 KD cells. F and G, DJ-1 deficiency intensified the formation of insoluble MAP1b-LC in vivo. F, The wild type or DJ-1 KO mice brain lysates were separated into Triton X-100 soluble or insoluble fractions. Western blot was used to analysis of MAP1b-LC distribution in the soluble or insoluble fractions. G, Quantification of relative levels of MAP1b-LC (*, p = 0.03).