Fig. 2

Degradation and poly-ubiquitination of Ezh1β are mainly contributed by NEDD4. a CHX chasing assay was performed in scramble/Ezh1β-FH and NEDD4 KD/Ezh1β-FH cell lines stressed with H₂O₂. scramble/Ezh1β-FH and NEDD4 KD/Ezh1β-FH cell lines were treated with 100 μM H₂O₂ for 24 h. During last hour of H₂O₂ treatment, 100 μg/ml cycloheximide (CHX) was added at indicated time points. Total proteins were extracted and immunoblot analysis was performed using anti-NEDD4 and anti-HA, anti-actin was used as loading control. b Quantification of remaining Ezh1β-FH protein percentage in a. Relative Ezh1β-FH was quantified in comparison remaining Ezh1β-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. c Poly-ubiquitination profile of Ezh1β under scramble and NEDD4 knock-down stable cell line. Scramble/1β-FH and NEDD4 KD/1β-FH indicate stable cell lines: scramble or NEDD4 KD constitutively expressing 1β-FH, respectively. Protein extracts were immunoprecipitated with FLAG and HA agarose beads and purified and ubiquitinated substrates were detected using anti-HA and anti-ubiquitin, respectively. Both scramble/1β-FH and NEDD4 KD/1β-FH stable cell lines were treated without or with H₂O₂ were indicated as MT and H₂O₂. 10 μM MG-132 was treated for 4 h before protein extraction. Ponceau S staining was used as loading control