Monoclonal mouse anti ha

Yeast protein extracts were prepared for western blotting as previously described47 (link). Samples resolved by SDS-PAGE were transferred to nitrocellulose membranes and blotted using the following antibodies: monoclonal mouse anti-HA (1:2000 dilution, ThermoFisher, 32–6700) and HRP-conjugated horse anti-mouse IgG (1:2000 dilution, Cell Signaling, 7076S). Western blots were developed with Supersignal West Pico Chemiluminescent Substrate (Pierce). To monitor the acquisition of N-linked glycosylation, whole cell protein extracts were digested with 1 U of Endoglycosidase H (New England Biolabs) overnight at 37 °C. Samples were resolved by SDS-PAGE and used for western blotting, as described above.

The eluates from coimmunoprecipitation using Dnj4HA as bait and 15 μg of protein from whole-cell lysate were subjected to electrophoresis in each well of a 15% SDS-PAGE experiment with subsequent transfer of proteins onto a polyvinylidene difluoride membrane (GE Healthcare, Boston, MA) using wet transfer at 70 V for 3 h. Membranes were blocked in TBST with 5% skim milk and incubated with 1:10,000 monoclonal mouse anti-HA (cat no. 26183; Thermo Fisher) or 1:5,000 rabbit anti-acetyl histone 3 (cat no. 06-942) as primary antibodies followed by 1:5,000 goat anti-mouse horseradish peroxidase (HRP) (cat no. 170-6516; Bio-Rad, Hercules, CA) or 1:5,000 goat anti-rabbit HRP (cat no. 170-6515; Bio-Rad). All immunoblots were visualized using chemiluminescence (GE Healthcare).