Fig. 5

Ezh1β Serine 560 phosphorylation is required for interaction between Ezh1β and NEDD4. a Poly-ubiquitination profiles of Ezh1β-T7, Ezh1βS560A-T7 and Ezh1βS560D-T7 under normal and oxidative stress conditions. HA-ubiquitin/Ezh1β-T7, HA-ubiquitin/Ezh1βS560A-T7 and HA-ubiquitin/Ezh1βS560D-T7 indicate C2C12 cell lines co-expressing HA-Ubiquitin and different forms of Ezh1β-T7 fusion proteins. Total proteins were extracted from these indicated stable cell lines. Ubiquitinated total proteins and ubiquitinated different Ezh1β forms were immunoprecipitated with HA agarose beads. Samples were eluted with HA peptide and running SDS-PAGE, anti-HA and anti-Ezh1β were used to detect total ubiquitinated proteins and ubiquitinated Ezh1β or mutant Ezh1β forms. 100 μM MG-132 was added and treated for 4 h before protein extraction. Ponceau S staining was used as loading control. b Interaction between NEDD4 and different forms of Ezh1β: Ezh1β, Ezh1βS560A and Ezh1βS560D. Cytosolic proteins were extracted and tandemly immunoprecipitated with Flag and HA agarose beads. NEDD4 antibody was used for immunoblotting analysis. Ponceau S staining was used as loading control