Fig. 1

Immunoprecipitation of Ezh1β-associated protein partners under oxidative stress condition. a Ezh1β-FH-associated protein complexes were tandemly affinity purified from cytosolic extracts of C2C12 stable cell line which expresses C-terminally FLAG-HA tagged Ezh1β. Flag and HA elutes indicate samples eluted with FLAG and HA peptides, respectively. Flag and HA elute samples were separated by SDS-PAGE and silver stained. Cytosolic protein extracts from C2C12 wild-type cell line were used as mock. Ezh1β-FH expressing C2C12 and normal C2C12 myotube samples, after changing differentiation medium for 2 days, were challenged with 100 μM H₂O₂ for 24 h. b Tandem affinity enriched Ezh1β-FLAG-HA associated proteins were identified by MS analysis. For each annotated protein, number of unique peptides and MASCOT score were listed. Three independent biological replicates data are presented. c Interaction among Ezh1β and ubiquitin E3 ligase: HUWE1, NEDD4 and FBXW8 were validated through co-immunoprecipitation (Co-IP) assay. Ezh1β-FH was immunoprecipitated from cytosolic extracts of two independent C2C12 stable cell line expressing Ezh1β-FH (#1 and #2). Wild-type C2C12 cell line was used as mock control. Ezh1β-FH expressing C2C12 and normal C2C12 myotube samples, after changing differentiation medium for 2 days, were challenged with 100 μM H₂O₂ for 24 h. Immunoblot analysis were performed with anti-HUWE1, anti-NEDD4, anti-CUL7, anti-FBXW8 and anti-HA