Immunoprecipitation assays were carried out as described previously [62 (link)]. The indicated cell lysates were hatched with specific primary antibodies and Protein A + G magnetic beads (MedChemExpress, Monmouth Junction, NJ, USA) overnight at 4 °C. Finally, after being washed with a lysis buffer, the immunocomplexes were analyzed via immunoblotting using the corresponding antibodies.
For ubiquitination analysis, cells were transfected with the specified plasmid and treated with MG132 (MedChemExpress) before treatment with lysis buffer. Immunoprecipitation was performed on cell lysates using the indicated primary antibodies. Then, the ubiquitinated proteins were hatched with protein A/G magnetic beads and detected via immunoblotting using specific primary antibodies.
For ubiquitination analysis, cells were transfected with the specified plasmid and treated with MG132 (MedChemExpress) before treatment with lysis buffer. Immunoprecipitation was performed on cell lysates using the indicated primary antibodies. Then, the ubiquitinated proteins were hatched with protein A/G magnetic beads and detected via immunoblotting using specific primary antibodies.
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