Fig. 1: HK2 can be SUMOylated at Lys 315 and Lys 492, and de-SUMOylated by SENP1.
a Coimmunoprecipitation of HK2 and UBC9 protein in HEK293T cells transfected with Flag-Ubc9 and HK2-HA plasmids. b SUMO1 and SUMO2 ligation with HK2 protein in HEK293T cells transfected with HK2-HA, UBC9, and Flag-SUMO1/Flag-SUMO2. c Endogenous HK2 SUMOylation was detected by immunoprecipitation with IgG or anti-HK2 antibody and then western blotting with anti-SUMO1 or anti-SUMO2/3 antibodies. d Sequence alignment of HK2 homologs in various species. The potential SUMOylation site is denoted in red. e 293T cells were transfected with HA-tag HK2 wild-type (WT), HK2-K315R, HK2-K492R, or HK2-DKR with or without His-SUMO1 and UBC9. Cell lysates were prepared for precipitation with Ni2+-NTA resin, followed by western blotting with indicated antibodies. f A reciprocal immunoprecipitation assay was performed with IgG or SUMO1 antibody for the lysates of PC3 cells, and then western blotting with HK1 and HK2 antibodies. g SENP1 depletion enhanced HK2 SUMOylation. UBC9, SENP1, SENP2, or SENP3 was stably knocked down by shRNA in PC3 cells. Cell lysates were prepared for precipitation with HK2 antibody-conjugated protein A/G agarose beads, and SUMOylation was detected by SUMO1 antibody. h Coimmunoprecipitation of HK2 and SENP1 protein in HEK293T cells transfected with Flag-SENP1 and HK2-HA plasmids. i Overexpression SENP1 decreased while UBC9 increased SUMOylation of HK2. Flag-UBC9 or Flag-SENP1 was transfected into PC3 cells, followed by detection of HK2 SUMOylated bands with SUMO1 antibody. Source data are provided as a Source Data file.
