Figure 5

SUMOylation of Grb2 increases its binding with Sos1. (A-B) HEK293T cells were co-transfected with the indicated plasmids. 24 hours later, cells were subjected to serum deprivation for 20 hours, followed the treatments with or without 100 ng/ml of EGF (A) or 10% serum (B) for the indicated time. Cell lysates were immunoprecipitated and subsequently immunoblotted with indicated antibodies. (C-D) HEK293T cells were co-transfected with the indicated plasmids and harvested after 48 hours. Cell lysates were immunoprecipitated and subsequently immunoblotted with the indicated antibodies. Densitometric readings were obtained with ImageJ V1.45 software. (E-F) Sos1 was not SUMOylated. HEK293T cells were co-transfected with the indicated plasmids and analyzed for SUMOylation by Ni²⁺-NTA pulldown (E) or immunoprecipitation (F). SUMOylated Grb2 was used as a positive control of SUMOylation, and mouse IgG as a negative control in immunoprecipitation.