Fig. 1

PDGFRα is SUMOylated. (a) COS-7 cells were co-transfected with indicated plasmids, followed by serum-starvation for 24 h. Cells were stimulated with 20 ng/ml PDGF-AA or PDGF-BB for different time periods, lysed and subjected to immunoprecipitation using an antibody against PDGFRα, followed by immunoblotting using anti-HA and anti-PDGFRα antibodies. Non-immune IgG was used as a negative control. Total cell lysates (TCL) were immunoblotted with an anti-PDGFRα antibody. HP95 (Alix) was used as a loading control. (b) Quantification of SUMOylated PDGFRα in panel A after different times of stimulation of PDGF-AA. Four independent experiments were performed and the standard deviation is indicated. (c) PAE cells with stably transfected PDGFRα were stimulated with 20 ng/ml PDGF-AA for 0, 1 and 4 h after serum-starvation for 24 h. Immunoprecipitation of SUMO1 was performed and SUMOylation of PDGFRα was determined by immunoblotting with a PDGFRα antibody. (d) RPE1 cells were stimulated with 20 ng/ml PDGF-AA for 0 and 45 min after serum-starvation or the presence of 10% FBS for 24 h. After immunoprecipitation with a SUMO1 antibody, samples were subjected to immunoblotting with a PDGFRα antibody. Receptor expression and loading control (Alix) were determined by immunoblotting of total cell lysates. (e) RPE1 cells, starved or not, were treated as in panel d, and cell lysates were heated at 95℃ and sonicated before immunoprecipitation with a SUMO1 antibody, followed by immunoblotting with antibodies against PDGFRα and PDGFRβ. (f) COS-7 cells transfected with PDGFRα and HA-SUMO1 were serum-starved and treated with 20 µM ginkgolic acid for 24 h before stimulation with PDGF-AA. SUMO1 was precipitated with HA antibody and PDGFRα was determined by immunoblotting. All experiments were performed three times or more except for the experiments shown in panels c and f which were performed two times. The immunoblots were cropped for clarity. Full length blots are presented in Figure S1