Fig. 2: Smurf1 regulates RNF220 protein stability.

A, B RNF220 protein was destabilized by overexpression of wild-type Smurf1 (A), but not their E3 ubiquitin ligase-defective mutants. Flag-tagged RNF220, myc-tagged wild-type or ligase-defective Smurf1 plasmids were transfected into HEK293 cells as indicated. After 48 h, cell lysates were analyzed by Western Blot. B Bar graphs, overlaid with the actual data points, show relative RNF220 protein expression (mean ± SD) normalized against the corresponding α-Tubulin. The control was set to 1. C, D Western Blot assays showing the protein level of RNF220 when co-expressed with Smurf1 in presence of MG132 or not. D Bar graphs, overlaid with the actual data points, show relative RNF220 protein expression (mean ± SD) normalized against the corresponding α-Tubulin. The respective controls were set to 1. E, F Effects of wild-type or ligase-defective Smurf1 or Smurf2 overexpression on the protein stability of RNF220. HEK293 cells were transiently transfected with the indicated plasmids. At 48 h posttransfection, cycloheximide was added to all samples, and cells were then harvested at the time points indicated. Level of endogenous RNF220 was determined by Western Blot with an anti-RNF220 antibody. In all cases, α-Tubulin was used as a loading control. The relative levels of RNF220 were quantified densitometrically and normalized against α-Tubulin. The data in F are the average of three independent experiments. G, H Western Blot results showing the effects of Smurf1 or Smurf2 knockdown on RNF220 protein level in HEK293 cells. HEK293 cells were transfected with the indicated siRNAs, and 72 h later, cells were harvested for Western Blot analysis. H Bar graphs, overlaid with the actual data points, show the relative expression (mean ± SD) of RNF220, normalized against the corresponding α-Tubulin. The control was set to 1. I, J Western Blot analysis showing the protein level of endogenous RNF220 when wild-type or E3 ubiquitin ligase activity defective Smurf1 was co-expressed with siRNAs against Smurf1 in HEK293 cells. The statistics of the result was shown in (J) with α-Tubulin as a loading control, and the control was set to 1. K, L Effect of Smurf1 knockdown on the protein stability of endogenous RNF220 in HEK293 cells. Cells were transiently transfected with the indicated siRNAs. At 72 h posttransfection, cycloheximide was added to all samples, and the cells were then harvested at the time points indicated. Protein level of RNF220 was determined by Western Blot with an anti-RNF220 antibody. The relative levels of RNF220 were quantified densitometrically and normalized against α-Tubulin. L The statistics showing the average of three independent experiments. IB immunoblot, WT wild-type, CA Smurf1 E3 ubiquitin ligase-defective mutant, CG Smurf2 E3 ubiquitin ligase-defective mutant, NC negative control, Chx cycloheximide, ns not significant. p > 0.05, *p < 0.05, **p < 0.01.