Ubiquitination assays and immunoprecipitation

Cells were treated with 10 μm MG132 (SELLECK, USA) for 6 h, and then the whole-cell lysates were extracted for immunoprecipitation (IP) to examine protein ubiquitination. The IP assay was carried out with the Pierce Co-IP Kit (Thermo Scientic) according to the manufacturer's protocol ²³. 8 μg of the affinity-purified ubiquitin or TβRI antibody were coupled with the resin at room temperature for 2 h. 500 μg of whole-cell lysates were mixed with the resin and the prepared appropriate experimental controls, incubating with gentle mixing for overnight at 4°C. The eluted complex samples were analyzed by Western blot.