Western blot analysis in SCA15 patients.
EBV immortalized lymphoblasts from three affected family members who carry the deletion and one family member without the mutation were used as a readily accessible source of protein; all samples came from members of family AUS1. Protein extraction was performed using lysis buffer containing 1× TBS, 1% Triton X-100, and a cocktail of protease inhibitors (Roche) with overnight lysis at −80 °C. Homogenates were diluted appropriately, mixed with 4× reducing sample buffer, and loaded onto 4%–12% precast gradient gels (NuPAGE, Invitrogen) for SDS-PAGE and immunoblotting. The antibodies to ITPR1 (1:1,000) and ACTB (1:5,000) were used as recommended by manufacturers.