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. 2019 May 28;116(24):11754–11763. doi: 10.1073/pnas.1820990116

Fig. 3.

Fig. 3.

Contributions of multiple E3 ligases to the control of HDM2 stability. (A and B) FBXO22 interacts with HDM2 in cells. (A) HEK293T cells were transfected with expression vectors as indicated. Extract proteins (3 mg) were used for immunoprecipitation. (B) HeLa or BT-549 cells were treated with MG132 (10 μM) for 6 h before lysis. Extract proteins (10 mg) were used for immunoprecipitation. (C) Immunofluorescence. MDA-MB-231 cells were used for immunofluorescence analysis as described in SI Appendix, Methods. (D) FBXO22 interacts with HDM2, p53, and KDM4A. MDA-MB-231 cells were transfected with a vector expressing HA-FBXO22, and the cells were treated with MG132 (10 μM) for 6 h before lysis. Extract proteins (5 mg) were used for immunoprecipitation as described in SI Appendix, Methods. (E) Effects of various E3 KDs in a panel of tumor cell lines. Immunoblot analysis of the relative protein level of FBXO22 and HDM2 in indicated cell lines treated with siRNA against various E3s. HDM2 quantification is shown below each blot image.