Fig. 1: RNF220 interacts with Smurf1 and Smurf2.
A, B Smurf1 (A) or Smurf2 (B) was co-immunoprecipitated with RNF220. C, D RNF220 was co-immunoprecipitated with Smurf1 (C) or Smurf2 (D). HEK293 cells were transiently transfected with different combinations of expression vectors of Smurf1, Smurf2, and RNF220, as indicated. Cell lysates were incubated with anti-Flag beads, washed, and subsequently analyzed by Western Blot. E, F Endogenous RNF220 could be immunoprecipitated by Smurf1 (E) or Smurf2 (F). Cell extracts of HEK293 cells were immunoprecipitated with antibody against Smurf1 or Smurf2, and endogenous RNF220 was detected by an anti-RNF220 antibody, with IgG as a negative control. G Endogenous Smurf1 and Smurf2 could be immunoprecipitated by RNF220. Cell extracts of HEK293 cells stably transfected with RNF220-Flag vectors were immunoprecipitated with anti-Flag beads, and endogenous Smurf1 or Smurf2 was detected by an anti-Smurf1 or anti-Smurf2 antibody, with IgG as a negative control. H–L The ability of different RNF220 deletion constructs or mutants to pull down Smurf1 (I, K) or Smurf2 (J, L) in co-immuniprecipitation assays. Schematic representation of the structures of truncated or mutated mouse RNF220 with amino acid numbers indicated is shown in (H). IB immunoblot, IP immunoprecipitation, WCL whole cell lysate, WT wild-type, FL full-length, SA RN220S282/286A mutant, and KR RNF220K321/323/326R mutant.
