Figure 3.

Identification and verification of SENP3-interacting proteins with SENP3–BirA   (A) Silver staining of precipitations by BioID. LO2 cells were stably transfected with pCDH–SENP3–BirA (named LO2–SENP3–BirA). LO2–SENP3–BirA cells were untreated or treated with DEN. (B) A pie chart showing the number of proteins identified by MS. (C) The interaction of SENP3 with TKT, NPM1, and ATP5F1A was verified by co-IP. LO2 cells were transfected with pCDH–SENP3–BirA and treated with DEN. (D) The in vitro interaction of SENP3 with TKT was validated by GST pull-down assay. Flag-SENP3 was transfected into HEK293T cells, and SENP3 was purified from cell lysates with anti-Flag M2 gels after 24 h of transfection. GST–TKT was obtained from prokaryotic expression system. (E) The physical interaction of SENP3 with TKT or ATP5F1A was measured by FRET with acceptor photobleaching method. After treatment with DEN for 2 h, LO2 cells were fixed and stained with primary antibodies and fluorescence-conjugated secondary antibodies. Scale bar: 10 μm. The efficiency of FRET from 10 cells are shown. (F) Transient SUMOylated proteins that interact with SENP3 were evaluated. co-IP was performed in HEK293T cells transfected with HA–SENP3.