Figure 1
From: SUMOylation of Grb2 enhances the ERK activity by increasing its binding with Sos1
Grb2 is SUMOylated in vivo and in vitro . (A) HEK293T cells were transfected with Flag-Grb2 along with or without His-SUMO1 and HA-Ubc9. SUMOylated proteins were purified from cell lysates using Ni2+-NTA affinity pulldown (Ni-pulldown) and SUMOylated Grb2 was detected by immunoblotting for the Flag tag. (B) His-SUMO1-conjugated proteins were purified by Ni-pulldown from HEK293T cells expressing Flag-Grb2, His-SUMO1, HA-Ubc9 or SENP1 and probed for Grb2. *indicates endogenous SUMOylated Grb2. (C) HEK293T cells were co-transfected with HA-SUMO1 and Flag-Ubc9. Lysates were immunoprecipitated with anti-HA(m) antibody or mouse IgG (as a control), and then immunoblotted with anti-Grb2 antibody. (D) The pE1E2S1 plasmid was co-transformed with pGEX4T1 (GST) or pGEX4T1-Grb2 (GST-Grb2) into E.coli BL21(DE3). Proteins were purified with GST agarose beads followed by Western blotting analysis.