Fig. 7: ISGylation delays proteosomal protein degradation in SCA1.

a Western blot of whole cerebellum reveals increase of ISGylated proteins in mutant Atxn1-KI mice, which changed during aging (left panel). Red and blue arrows indicate 40 kDa and 100 kDa bands that were changed significantly in mutant Atxn1-KI mice. The same filter was reprobed with anti-Atxn1 and anti-Ub antibodies (right panels). Blue and red arrows indicate 100 and 40 kDa bands, respectively. Black arrows indicate the ISG15 monomer, whose signal remained after washing for reprobing. Anti-GAPDH served as loading control. b Reprobing of the same filter with anti-Atxn1 and anti-Ub antibodies. The 100 kDa (blue arrow) and 40 kDa (red arrow) bands were reactive to both antibodies. Small and large black arrowheads indicate residual normal and mutant Atxn1 proteins before degradation. Larger magnified images (right panels) show the bands of normal and mutant Atxn1. c Possible patterns of Atxn1 protein modification by both ISG15 and Ub. d Western blotting with anti-Ub and anti-Atxn1 antibodies revealed that ISGylated proteins immunoprecipitated from whole cerebellum sample of mutant Atxn1-KI mice included multiple forms of ubiquitinated Atxn1. Thin blue arrows indicate monomeric Atxn1 that is both ISGylated and ubiquitinated. Thick blue arrows and blue arrowheads indicate conjugated Atxn1 via cross-linking by transglutaminases. These high molecular cross-linked Atxn1 species were both ISGylated and ubiquitinated. e Super-resolution microscopy of Purkinje cells in Atxn1-KI and normal sibling mice at 4 weeks of age. f The protocol for the pulse-chase assay to examine the effect of ISGylation on Ub-proteasome-dependent and autophagy-dependent protein degradation. The lower panel shows the result of the pulse-chase assay. g The pulse-chase assay shows protein degradation after protein synthesis is blocked by cycloheximide. Degradation of Myc-Atxn1-86Q was promoted by siRNA-ISG15, while that of FLAG-Ku70 or Myc-Atxn1-33Q was not.