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. 2016 Jul 8;7(31):50522–50534. doi: 10.18632/oncotarget.10494

Figure 1. Modification of PES1 by SUMO-1, SUMO-2 and SUMO-3.

Figure 1

A. COS-7 cells were co-transfected with Myc-SUMO-1, −2 or −3 and GPF-tagged PES1, and then subjected to western blot with anti-GFP antibody. B. COS-7 cells were transfected with Flag-tagged PES1 and Myc-tagged SUMO1 and then subjected to immunoprecipitation with anti-Flag antibody followed by western blot with anti-Myc antibody. C. COS-7 cells were cotransfected with Flag-tagged PES1 and GFP-tagged SUMO1 or SUMO1/GA and then subjected to immunoprecipitation with anti-Flag antibody, followed by western blot with anti-GFP antibody. D. COS-7 cells were transfected with GFP-PES1 alone, GFP-PES1 plus Myc-SUMO1 without or with UBC9, and then subjected to immunoprecipitation with anti-GFP antibody followed by western blot with anti-Myc or anti-Flag antibody. E. In vitro SUMOylation assay was performed using a SUMOylation kit (Enzo Life Sciences). Affinity-purified GST or GST-PES1 was incubated with SAE1-SAE2 (E1), UBC9 (E2), Mg-ATP and SUMO-1, as indicated at 30°C for 60 min, and then analyzed by immunoblotting. F. MCF-7 cells were subjected to serum starvation for 3 days, followed by treatment with 10 nM E2 for 4 h, and the cell extract was then subjected to immunoprecipitation with anti-PES1 antibody or anti-immunoglobulin G, followed by western blot analysis with anti-SUMO1 or anti-PES1 antibody. G. MCF-7 cells were transfected with Myc-SUMO1 for 24 h, and the cells were then subjected to serum starvation for 3 days, followed by treatment with 10 nM E2 for 4 h. Cell extract was prepared and subjected to immunoprecipitation with anti-PES1 antibody or anti-immunoglobulin G, followed by western blot analysis with anti-Myc or anti-PES1 antibody. H. MCF-7 cells were starved for 3 days, followed by treatment with 10 nM E2 for different periods, and then subjected to western blot analysis with anti-PES1 antibody.