Western Blot Analysis of Frozen Tissues

Fresh frozen tissue lysis and Western blotting (WB) were performed as previously described [14 (link)]. The primary antibodies are provided in Additional file 1: Table S1, and many were previously tested in autopsy tissue [14 (link)]. WBs for each antibody were repeated at least twice, with similar results. The densitometric analysis was performed by scanning the X-ray films with optimal exposures on a ChemiDoc™ Touch imager (Bio-Rad, Hercules, CA). The bands were quantified by using Image Lab 6.0 software (Bio-Rad). Individual protein values were normalized to the corresponding actin or IDH1-R132H values, except for phosphoprotein values that were normalized to the corresponding unphosphorylated protein values. Minus values were manually adjusted as zero. Results were expressed as percent of the highest normalized values.