Anti dpm1

Anti-HA and anti-Ubiquitin were purchased from Roche and BioMol, respectively. Anti-Sec61 antibody was a generous gift from Jeffrey L. Brodsky (University of Pittsburgh, Pittsburgh, PA). Anti-Kex2 and anti-Pma1 antibodies were purchased from Abcam. Anti-Pep12, anti-Dpm1, anti-Pho8, and anti-Pgk1 antibodies were purchased from Invitrogen. Immunoblots were incubated with the indicated primary antibodies and appropriate HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Sigma). The HRP-chemiluminescent signal was visualized by enhanced chemiluminescence (Pierce, USA) or Luminata Forte Western HRP substrate (Millipore).

Western blotting was performed with 0.2 OD₆₀₀ units of cells harvested for sample preparation. Frozen cells were treated with 10% trichloroacetic acid (TCA) for 5 min on ice. After TCA was discarded by centrifugation, the pellet was washed with ice-cold acetone and resuspended in 1 × sample buffer (75 mM Tris-HCl pH 6.5, 10% glycerol, 25 mM DTT, and 0.6% SDS). The samples were homogenized using 0.5 mm zirconia beads (Yasui Kikai, YZB05) and FastPrep-24 (MP Biomedicals, 6004-500) with a setting of 60 s at 6.0 m/s, and then incubated at 65˚C for 10 min. Samples were separated by SDS-PAGE followed by Western blotting. Anti-FLAG (Sigma–Aldrich, F3165, 1:1000), anti-Pho8 (Abcam, ab113688, 1:1000), anti-Ape1 (1:5000)^{66 (link)}, anti-β-actin (Wako, 010-27841, 1:500), anti-Dpm1 (Invitrogen, A6429, 1:1000), anti-Gsp1 (ImmuQuest, IQ241, 1:10000), anti-Van1 (a gift from Koji Yoda, 1:3000), and anti-GFP (Roche, 11814460001, 1:1000) were used as primary antibodies. Chemiluminescence was raised by Femtoglow HRP Substrate (Michigan Diagnostics, 21008) and blots were visualized using LAS-4000 (GE Healthcare) or FUSION-FX7 (Vilber-Lourmat) imaging systems.