Fig. 1

hSSB1 is SUMOylated by SUMO3. a Forty-eight hours after HEK293T cells were cotransfected with FLAG-hSSB1 and HA-SUMO1-3, the cells were lysed and the proteins analyzed by western blotting or immunoprecipitation (IP) using an anti-FLAG antibody followed by western blotting. WCL whole cell lysate. b Forty-eight hours after HEK293T cells were cotransfected with FLAG-hSSB1 and His-SUMO3, the cells were lysed. After sonication, the lysates were incubated with nickel-nitrilotriacetic acid (Ni-NTA) beads, and the pulled down proteins were analyzed by western blotting. c HEK293T cells treated with or without etoposide were lysed in RIPA-SDS lysis buffer, immunoprecipitated with SUMO2/3 affinity beads and the proteins, analyzed by western blotting. d Twenty-four hours after HEK293T cells were cotransfected with FLAG-hSSB1 and HA-SUMO3, the cells were treated with or without 100 μM etoposide, as indicated, for 24 h, and then the cells were lysed and the proteins analyzed as in a. e HEK293T cells cotransfected with FLAG-hSSB1 and HA-SUMO3 were treated with etoposide for the indicated times before harvesting, 48 h post transfection, and then the cells were lysed and the proteins analyzed as in a. f HEK293T cells with stably knocked out UBC9 were cotransfected with FLAG-hSSB1 and HA-SUMO3 for 24 h, and then treated with 100 μM etoposide for 24 h, as shown in a. g, h HEK293T cells were transfected with the indicated FLAG-hSSB1 plasmids with HA-SUMO3 and, after 24 h, were treated with 100 μM etoposide, as indicated, for 24 h. Then, the cells were lysed and the proteins analyzed by western blotting or IP using the anti-FLAG antibody followed by western blotting