Western Blot Analysis of SUMOylated Proteins

Cells were harvested as we have described [13 (link), 33 (link), 34 (link)] except for experiments to measure sumoylation where iodoacetemide (10 mM) was added to the lysis and wash buffers to preserve SUMOylation. Western blots were performed as described [33 (link), 35 , 36 (link)]. Briefly, 50 μg of protein was resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary antibody (1/1000 dilution) and secondary antibody (1/10000 dilution). Bound antibody was detected with the LI-COR system. Blots were incubated with IRDye® 680RD Goat Anti-Mouse Li-COR dyes and visualized with an Odyssey® CLx Imaging System (LI-COR, Inc., Lincoln, NE) using LI-COR Odyssey software. Band intensities were quantified using the Quantity One software (Bio-Rad, Hercules CA) and intensities normalized to loading control as previously described [34 (link)].