Fig. 4

Ezh1β Serine 560 phosphorylation is required for Ezh1β poly-ubiquitination and degradation upon oxidative stress. a Mass spectra showing phosphorylation site of Ezh1β localized at Serine 560 amino acid. b Sequence alignment between C terminal of Ezh1α and Ezh1β protein sequence. Consensus sequence was highlighted. c LC–MS/MS quantification of Ezh1βS560 phosphorylation levels under normal and oxidative stress conditions, p-value = 0.001702. d Increased Ezh1β Serine 560 phosphorylation status was validated through Immunoprecipitation of Ezh1β-FH coupled with immunoblotting through anti-HA, anti-p-Ser, respectively. Ponceau S staining was used as loading control. e Quantification of phosphorylated Ezh1β Serine 560 level presented in d, p-value = 0.0005. CHX chasing assay of Ezh1β-FH, Ezh1βS560D-FH (f) and Ezh1βS560A-FH (g) under normal and oxidative stress conditions. Ezh1β-FH, Ezh1βS560D-FH and Ezh1βS560A-FH mean different stable cell lines expressing wild-type Ezh1β, point mutation form Ezh1βS560D and Ezh1βS560A fusion with FLAG and HA. Total proteins were extracted from indicated stable cell lines under normal and oxidative stress conditions. 100 mg/ml CHX was added during last hour before protein extraction and treated as indicated time points. Immunoblotting was used to detect remaining protein levels of Ezh1β or Ezh1β mutant forms with anti-HA. Anti-actin was used as loading control. h–k Quantification of remaining Ezh1β-FH, Ezh1βS560D-FH (h, i) and Ezh1βS560A-FH (j, k) shown in f and g, respectively. MT means myotube stage sample and H₂O₂ means myotube sample stressed with 100 μM H₂O₂ for 24 h. Actin protein abundance was normalized and data were expressed as mean ± SD from three biological replicates. Values above each bar indicate Student’s t-test p value