Fig. 6

HUWE1-mediated changes of Ezh1α through regulating diverse poly-ubiquitination profiles of Ezh1α under normal and oxidative stress conditions. a Ezh1α-FH-associated protein complexes were tandemly affinity purified from nuclear extracts of C2C12 stable cell line which expresses C-terminally FLAG-HA tagged Ezh1α. 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. Nuclear protein extracts from C2C12 wild-type cell line were used as mock. Ezh1α-FH expressing C2C12 and normal C2C12 myotube samples, after changing differentiation medium for 2 days were used for nuclear protein extraction. b Tandem affinity enriched Ezh1α-FLAG-HA associated proteins were identified by MS analysis. For each annotated protein, number of unique peptides and MASCOT score were listed. Data from at least three independent biological replicates data are presented. c Interaction among Ezh1α and SUZ12 ubiquitin E3 ligase HUWE1 were validated through Co-immunoprecipitation (Co-IP) assay. Ezh1α-FH was immunoprecipitated from nuclear extracts of two independent C2C12 stable cell line expressing Ezh1α-FH (#1 and #2). Wild-type C2C12 cell line was used as mock control. Ezh1α-FH expressing C2C12 and normal C2C12 myotube samples, after changing differentiation medium for 2 days, were used for protein extraction. Immunoblot analyses were performed with anti-HUWE1, anti-SUZ12, and anti-HA. d CHX chasing assay were performed in scramble/Ezh1α-FH and HUWE1 KD/Ezh1α-FH cell lines under normal myotube samples. 100 μg/ml cycloheximide (CHX) was added at indicated time points. Total proteins were extracted and immunoblot analysis was performed using anti-HUWE1 and anti-HA, anti-actin was used as loading control. e Quantification of remaining Ezh1α-FH protein percentage in d. Relative Ezh1α-FH was quantified in comparison remaining Ezh1α-FH with initial total protein amount at indicated CHX treatment time points. Data were expressed as mean ± SD from three biological replicates. ImageJ software was used to determine protein abundance. Values above each bar indicate Student’s t-test p value. f Dynamic interaction between HUWE1 and Ezh1α under normal and stress conditions. Ezh1α-FH was enriched with FLAG and HA agarose beads, then FLAG and HA peptide were used to elute immunoprecipitated protein partners. Interaction between HUWE1 and Ezh1α, ubiquitination profile of Ezh1α were determined by anti-HUWE1 and anti-ubiquitin. Ponceau S staining was used as loading control