Western blot analysis
A rabbit polyclonal antibody, anti-MagT1, was raised against the N-terminal domain of the final cleaved human MagT1 protein using a synthetic peptide, INFPAKGKPKRGDTYELQV (amino acid residues 140–158), coupled to keyhole limpet hemocyanin. Affinity-purified rabbit anti-human MagT1 antibody was diluted in TBS (Tris-buffered saline, 20 mM Tris, 200 mM NaCl, pH 7.6) containing 0.5 % BSA at a final concentration about 0.7 μl/ml. For subcellular fractionation, cells were suspended in lysis buffer (0.25 M sucrose, 10 mM triethanolamine-acetic acid pH 7.6, 1 mM EDTA) containing protease inhibitors (1 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin). Protein concentrations were determined using Bio-Rad protein assay reagent. SDS-PAGE was performed according to Laemmli. For immunobblotting, the proteins were electrophoretically transferred to polyvinylidene difluoride membranes (HybondR, Amersham Biosciences) by semidry electroblotting for 45 min. Western analysis was performed by incubating the blots with antiMagT1 antibody or anti-MagT1 antibody preabsorbed with 50 × antigen peptide (control for antibody specificity) overnight at 4EC followed by three washes with TBS/0.1% Tween-20, 10 min each. The blots were then incubated with 1/10,000 horseradish peroxidase-conjugated donkey anti-rabbit secondary (Sigma Aldrich) antibody for 1 h. After washing three times with TBS/Tween-20, 10 min each, the blots were visualized with ECL (Amersham Biosciences) according to the manufacturer's instructions.