Fig. 5
From: PHF20L1 antagonizes SOX2 proteolysis triggered by the MLL1/WDR5 complexes
PHF20L1 inhibits WDR5-dependent ubiquitination of SOX2. a Silencing of PHF20L1 antagonized the accumulations of SOX2 protein induced by knocking down the components of the MLL1/WDR5 complexes. PA-1 cells were co-transfected with siRNAs targeting WDR5, RBBP5, ASH2L, MLL1, or PHF20L1 respectively for 48 h, and the protein levels of SOX2 were examined by western blotting. The relative protein levels of SOX2 were quantified and plotted on the lower panels respectively. b PHF20L1 inhibited the ubiquitination of SOX2. 293 cells stably expressing GFP-SOX2 or GFP-SOX2-K42R mutant were co-transfected with plasmids encoding HA-ubiquitin, Flag (plasmid vector) or Flag-WDR5, and Flag-PHF20L1 for 48 h respectively. Cells were incubated with MG-132 for 6 h and SOX2 was immunoprecipitated using anti-SOX2 antibody. Ubiquitinated SOX2 was detected by an anti-HA antibody. The ubiquitinated SOX2 was densitometry quantified and plotted using Gel Image analysis software. a and b *p < 0.05, **p < 0.01, ***p < 0.001. The data were represented as mean ± SD. c SOX2 interacted with PHF20L1. Upper panel: endogenous SOX2 interacted with endogenous PHF20L1 in PA-1 cells. Bottom panel: exogenous SOX2 associated with exogenous PHF20L1 in 293 cells stably expressing GFP-SOX2. Co-immunoprecipitated SOX2 or Flag-PHF20L1 were examined with anti-Flag or SOX2 antibodies respectively. Normal rabbit serum (NRS) was taken as a negative control
