To evaluate Esc2 protein expression levels, 10 mL of log-phase cells (OD600nm ~ 1.0) were harvested and whole cell lysate was extracted using glass bead beating. The concentration of protein in cell lysates were normalized using the Bradford assay (Bio-Rad) and TAF-tagged Esc2 proteins were detected on western blot via chemiluminescence method. The antibodies used were a rabbit anti-Protein A primary antibody (1:10,000, Sigma) and a goat anti-Rabbit HRP secondary antibody (1:10,000).

Protein binding assays were performed using C-terminal 6xHIS tagged Ubc9 was expressed in BL21 cells and purified using a Ni-NTA affinity column. N-terminal Protein-A tagged Esc2 WT and Esc2-D430R mutant proteins were similarly expressed and purified with IgG sepharose resin. 40 μg of Esc2 (WT and D430R) was incubated and bound to 20 μL IgG resin; and was subsequently incubated with 100 μg of purified Ubc9 in a final volume of 200 μL for 2 hours on ice. The IgG resins were then washed 5 times with 1 mL PBS (Phosphate-Bu with 0.2% NP-40. After washing, the bound Ubc9 protein was eluted from the IgG resin with 20 μL of 0.1M glycine-HCl and then 20 μl 1% SDS loading buffer for visualization by Coomassie staining.

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