Fig. 2: Pramel15 interacts with DNMT1 and mediates DNMT1 degradation via ubiquitin-proteasome pathway.
From: Pramel15 facilitates zygotic nuclear DNMT1 degradation and DNA demethylation
a Flag-immunoprecipitation from 293FT cells transfected with Pramel15-HA and DNMT1-Flag (upper) or UHRF1-Flag (lower) expressing plasmids. b High-content images show DNMT1-GFP level with or without Pramel15-mCherry induction by doxycycline (Dox) treatment for 48 h (left) and the boxplot graph shows the statistic results of DMSO group (n = 3973) and Dox group (n = 3109) (right). Boxplots show median (center line), 25th to 75th percentiles (hinges). The whiskers go down to the smallest value and up to the largest. Two-sided t-tests assuming unequal variances were performed. c Western blotting of DNMT1 protein levels in T-REx293 cells expressing Dox-inducible Pramel15-Flag with or without 5 μM MG132 treatment. d Schematic representation of DNMT1 and its truncations. e, f Flag-immunoprecipitation from 293FT cells transfected with DNMT1-Flag or its truncations and Pramel15-HA expressing plasmids. Asterisk: heavy chain of antibody. g Western blotting of Flag-DNMT1 and Flag-DNMT1(Δ350-609aa) protein levels in 293FT cells with or without Pramel15-HA. h Western blotting of Flag-DNMT1(350-609aa) protein levels in mES cells expressing Flag-DNMT1(350-609aa) and Dox-inducible Pramel15-HA. i Flag-immunoprecipitation in 293FT cells transfected with plasmids expressing His-ubiquitin, Flag-DNMT1(1-609aa), and Pramel15-HA or blank plasmids treated with 20 μM MG132. All immunoprecipitation and quantification experiments analyzed by Western blotting were repeated at least three times independently.
