Denaturing Pulldowns of Hrd1

Denaturing pulldowns of Hrd1 were performed as described previously (Baldridge and Rapoport, 2016 (link)) with the following modifications. Cells with a centromeric plasmid bearing an endogenous Hrd1 promoter and a Hrd1-His10 were grown to mid-log phase and lysed in 50 mM HEPES pH 7.4, 300 mM KCl, protease inhibitors, 1 mM PMSF, 1.5 µM pepstatin, 8M urea (to prevent additional Hrd1 autoubiquitination), and 5 mM NEM (to inhibit deubiquitinating enzymes). The lysates were centrifuged at 2000 x g for 10 min, and the supernatant was collected and re-centrifuged for 30 min in a Ti45 rotor at 42,000 rpm (RCF_avg 138,001). The membranes were solubilized in 50 mM HEPES pH 7.4, 300 mM KCl, protease inhibitors, 1 mM PMSF, 6M urea, 1.5% Triton X-100 final and 25 mM imidazole) for 1 hr at 4°C. His-tag Dynabeads (Life Technologies) were added (0.25 mL per 1,500 OD cells) and incubated for an additional 1 hr. The beads were washed three times with a 30-fold excess buffer. Hrd1-His10 was eluted with buffer including 400 mM imidazole. The samples were analyzed by SDS-PAGE and immunoblotting with anti-Hrd1 and anti-ubiquitin antibodies (clone P4D1, Santa Cruz). Unbound IMAC flow-through was used for a loading control to demonstrate equal material input.