Fig. 2

sPRDM16 was SUMOylated by SUMO1 on lysine-568. a sPRDM16 was SUMOylated in vivo. HEK293T cells were transfected with the indicated plasmids for 36 h. Immunoprecipitation was performed with anti-FLAG M2 agarose beads. The immunoprecipitates (IP) and the original whole-cell lysates (WCL) were analyzed by immunoblotting (IB) with anti-HA or anti-FLAG antibodies. b SENP1 de-SUMOylated sPRDM16. HEK293T cells were transfected with HA-SUMO1, Flag-sPRDM16, RGS-SENP1, or RGS-SENP1m as indicated. Flag-sPRDM16 proteins were pulled down by anti-Flag M2 agarose beads from cell lysates. Bound proteins were blotted with anti-Flag. Cell lysate was immunoblotted (IB) with anti-Flag antibody, anti-HA antibody, or anti-RGS antibody. c K568 was the primary SUMOylation site of sPRDM16. HEK293T cells were transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-FLAG M2 agarose beads, followed by Western blot (WB) analysis using anti-HA or anti-FLAG antibodies