Figure 2. Shp2 is SUMOylated at multiple sites and K⁵⁹⁰ is a major SUMO-site.

(A) Schematic maps of Shp2 domains. N-SH2 (1–106 aa), N-terminal Src Homology 2 domain; C-SH2 (107–220 aa), C-terminal Src Homology 2 domain; ΔSH2: deletion of amino acids 1–209 containing N-SH2 and C-SH2 domain regions. (B–C) pEF-HA-N-SH2, -C-SH2 (B), or −ΔSH2 (C) were co-transfected with Flag-Ubc9 and His-SUMO1 into 293T cells, and then SUMOylation assays by using Ni²⁺-NTA agarose beads were performed as above. (D) 293T cells co-transfected with ΔSH2^WT or mutant ΔSH2^K590R with or without Flag-Ubc9 and His-SUMO1 were lysed and treated for SUMOylation assays. (E) 293T cells co-transfected with full-longth Shp2^WT or Shp2^K590R with or without Flag-Ubc9 and His-SUMO1 were lysed and treated for SUMOylation assays. (F) The GST-Shp2^WT or GST-Shp2^K590R plasmid was co-transformed with pE1E2SUMO1 into E.coli BL21 (DE3). Immunoblotting was conducted after GST pull down.