Fig. 6

Overexpression of A216P-GFP induces aggregation of PRPF31 protein. The ARPE-19 cell line was transfected with PRPF31-GFP (a-c, g-i) and A216P-GFP (d-f, j-l). The GFP-tagged proteins (green) and immunostained cells with anti-PRPF31 antibodies (red) are shown. PRPF31-GFP was found mainly in the cell nucleus (a-c; 1, g-i) and to a lesser extent, in the cytoplasm (a-c; 2). A216P-GFP transfected cells present PRPF31 aggregation in the cytoplasm (d-f; 3, j-l), and a very minor signal in the nucleus (d-f; 4). Images correspond to a maximum projection of a Z-stack. Western blot analysis of soluble and insoluble fractions of the transfected cells show a decrease in the concentration of endogenous PRPF31 in the detergent-soluble fraction and an increase in the detergent-insoluble fraction of the cells transfected with A216P-GFP. Anti-γ-tubulin antibody was used as loading control (m). Densitometry quantification of the blots (n) shows a significant increment of A216P-GFP protein in the detergent-insoluble fraction when compare with the among of A216P-GFP protein present in the soluble fraction (n). The boxplot n represents the ratio PRPF31/γ-tubulin in the soluble and insoluble fractions of PRPF31-GFP and A216P-GFP transfected ARPE-19 cells (n = 3 in each group). Statistically significant differences were determined by Mann-Whitney U-test (*p < 0.05). Western blot analysis of soluble and insoluble fractions of PRPF31-GFP and A216P-GFP transfected ARPE-19 cells and co-transfected with WT PRPF31 tagged to Flag showed a decrease in the concentration of WT PRPF31-Flag in the detergent-soluble fraction and an increase in the detergent-insoluble fraction of the cells co-expressing the mutant A216P-GFP protein. Anti-GAPDH antibody was used as loading control (o). Scale bars (a-f) represent 25 μm and (g-l) 10 μm