Fig 1: RNF4 acts as E3 ubiquitin ligase of SUMOylated CBX2. a Western blot of endogenous CBX2 following transfection of increasing amounts of wt FLAG/RNF4 or FLAG/RNF4-CS (0,5, 1 or 2 µg) in HEK293-FT cells. b IP of endogenous CBX2 upon FLAG/RNF4 overexpression in HEK293-FT cells. c IP of endogenous CBX2 following transfection of either His/SUMO2 (3 µg) or FLAG/RNF4 alone or in combination. Immunoblotting was performed with indicated antibodies. d Endogenous CBX2 polyubiquitination status K562 stably expressing shRNF4 construct #1 and shSCR control treated with 5 µM SAHA at 3 cells Total extracts were immunoprecipitated with anti-CBX2 antibody. CBX2 polyubiquitination rate in SAHA-treated shRNF4#1-transduced K562 cells compared to shSCR control are shown. e Western blot analysis of endogenous CBX2 in K562 cells stably expressing two different shRNA construct targeting shRNF4 (#1 and #2) or shSCR control. ERK1/2 was used as loading control
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