Immunoprecipitation and Western Blot

For IP and western blot, dynabeads m-270 Epoxy (Invitrogen) coupled with antibodies were prepared and then cell lysates were added, and the antibodies−lysate mixtures were rotated at 4 °C for 1 h. Immunocomplexes separated from the dynabeads were washed with lysis buffer and then suspended with SDS blue loading buffer. To detect ubiquitinated proteins, lysis was performed under 80 °C for 10 min. Western blot analysis was performed as we described previously.^{40 (link)} In brief, an equal amount of total protein extracted from cultured cells were separated by 12% SDS–PAGE and transferred to polyvinylidene difluoride membranes. The blots were blocked with 5% milk for 1 h. Primary Abs and horseradish peroxidase-conjugated secondary Abs were each incubated for 1 h. The bounded secondary antibodies were reacted to the ECL detection reagents and exposed to X-ray films (Kodak, Japan).