Anti gdap1

Total protein cell extracts were prepared using a cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) with 1 mM PMSF and Protease Inhibitor Cocktail 1/200 v/v (Sigma-Aldrich). Protein were separated by SDS-PAGE, transferred onto nitrocellulose membrane Amersham Protran (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and analyzed by standard Western blotting using rabbit polyclonal anti-GDAP1 (Abcam, Cambridge, MA, USA), purified mouse anti-GM130 (BD Transduction Laboratories, San Jose, CA, USA), rabbit polyclonal anti-B4GALT3 (Proteintech, Chicago, IL, USA), rabbit polyclonal anti-GORASP2 (Proteintech), rabbit anti-TGN46 (Sigma-Aldrich), mouse monoclonal anti-beta Actin (Proteintech) antibodies and secondary anti-rabbit IgG and anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibodies (Sigma-Aldrich) followed by enhanced chemiluminescence (Western Bright Sirius Advansta, San Jose, CA, USA).

Total protein cell extracts were prepared using a cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) with 1 mM PMSF and Protease Inhibitor Cocktail 1/200 v/v (Sigma-Aldrich, Saint Louis, MI, USA). Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane Amersham Protran (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and analyzed by Western blotting using rabbit polyclonal anti-GDAP1 (Abcam, Cambridge, MA, USA) and secondary anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody (Sigma-Aldrich, Saint Louis, MI, USA) followed by enhanced chemiluminescence (Western Bright Sirius Advansta, San Jose, CA, USA).

Yeast cells were grown overnight at 28 °C in SC -leu medium. Protein extracts were prepared after disrupting cells with acid-washed glass beads in 2 × urea electrophoresis sample buffer (50 mM Tris-HCl pH 6.8; 1.6% SDS; 7% glycerol; 0.016% bromophenol blue; 4% β-mercaptoethanol; 8M urea) supplemented with protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MI, USA). Samples were analyzed by standard Western blotting using rabbit polyclonal anti-GDAP1 (Abcam) or rabbit polyclonal anti-Histone H3 (Abcam) antibodies and secondary anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibodies (Sigma-Aldrich), followed by enhanced chemiluminescence (GE Healthcare, Boston, MA, USA).