The interaction between ID1 and RNF4 suppresses SP1 ubiquitination. (A) The silver nitrate staining results of the immunoprecipitation HA-Id1 interactome. (B-C) Both HA and FLAG coimmunoassays showed significant interactions between HA-ID1 and RNF4-FLAG. (D) ID1 and RNF4 interact with each other in vitro. Both purified GST-RNF4 and GST-ID1 pull-down assays showed significant interactions with His-ID1 and His-RNF4. (E) The levels of SP1 protein, but not ID1, were significantly reduced in HS-5 cells transduced with shRNA against RNF4. (F) ID1 inhibits the RNF4-SP1 interaction in vitro. Purified GST-RNF4, HA-ID1, and FLAG-SP1 were used for GST pull-down assays, and anti-GST, anti-HA, and anti-FLAG antibodies were used for western blotting. (G) SP1, HA-ID1, RNF4-FLAG, and MYC-ubiquitin were cotransfected into 293T cells. Anti-SP1 immunoprecipitation assays have shown that HA-ID1 significantly inhibits SP1 ubiquitination via RNF4. (H) Schematic representation of the ID1 and RNF4 truncations. (I) The anti-FLAG immunoprecipitation assay showed a significant RNF4-FLAG interaction with the ID1 C-terminal. (J) Anti-HA immunoprecipitation assay showed significant interaction of HA-ID1 with the RNF4 SIM domain. (K) HS-5 cells with stable expression of truncated ID1 were transiently transfected with either RNF4 or empty vectors. Western blot analysis of SP1, HA, and FLAG-related proteins. (L-M) MTT assays showed significant inhibition of the proliferation in Kasumi-1 and THP-1 cells cocultured with HS-5 cells expressing RNF-FLAG and HA-ID1 C del. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001.