Figure 2
From: SUMOylation of Grb2 enhances the ERK activity by increasing its binding with Sos1
K56is a critical site for Grb2 SUMOylation. (A) HEK293T cells were co-transfected with Flag-Grb2 wild-type or mutants along with or without His-SUMO1 and HA-Ubc9. The in vivo SUMOylation assay using Ni2+-NTA beads was conducted as described in Methods. (B) Amino acid sequence alignment of Grb2 partial sequences located between N-SH3 and SH2 domains from different species. The conserved lysine in the linker region (yellow) has been underlined. (C) HEK293T cells were transfected with Flag-Grb2WT or Flag-Grb2K56R plasmid along with the increased amount of His-SUMO1 and HA-Ubc9 plasmids. The levels of SUMOylation were determined as described in Methods. (D) The plasmid GST-Grb2 or GST-Grb2K56R was co-transformed with pE1E2S1 plasmid into BL21(DE3). Western blotting was conducted after GST pulldown.