Fig 2. SDS-PAGE allows the resolution of ATXN3 monomers, polymers/modified forms and HMW fibrils.

(A) HEK-239T were transfected with GFP-ATXN3 WT or GFP-ATXN3 polyQ plasmids at the indicated times cell were collected and subjected to the fractionation and SDS-PAGE as described in the protocols section. The complete gel including the stacking gel was transferred onto nitrocellulose followed by western blotting using a GFP antibody. In green and blue are indicated parts corresponding to the separating and stacking phases. (B) HEK-239T were transfected with ATXN3 polyQ and 7 days later treated with DMSO or MG132 (5 μM) overnight. Cells were then lysed and processed as in (A) (left panel). Quantification of the insoluble GFP-ATXN3 polyQ, in monomers (middle panel) and HMW aggregates (right panel) (n = 3). The relative ratio between the insoluble and soluble fraction was calculated and called insolubility index. The relative insolubility index of DMSO-treated samples is set to 1. Student’s t‐test was used to determine the statistical significance. Error bars represent the standard deviation. * p<0.5, ** p<0.01 and *** p<0.001.