Sumo 3

Transfected HEK 293 cells were collected and were divided into two. One half was used for the initial time point. The second half was incubated with 2 μM preformed chains of SUMO-2 or SUMO-3 (BostonBiochem) and de-sumoylation was allowed to proceed for 30 min 37 °C. All samples were denatured in 6X SDS loading buffer and boiled for 15 minutes. Cleavage of the poly-SUMO chains was assessed by Western blot analysis.

Bead conjugated or unconjugated pro-SUMO1, SUMO1, SUMO2 and SUMO3 were purchased from Boston Biochem. The Sumoylation kit for the productions and detection of sumoylated RG1 was purchased from Enzo Lifesciences (BML-UW8955-0001). Commercially available Ulp1 for in vitro desumoylation reactions was purchased from Thermo-Fisher (12588018). TCEP was purchased from Thermo Fisher (Pierce 120490). 10x Cell Lysis Buffer for cell extracts was purchased from Cell Signaling Technology (#9803) and cell extracts from mammalian cells were generated as recommended by the manufacturer. The anti GFP antibody used was JL-8 was from Takara (632381). The anti SUMO1 and SUMO2/3 antibodies were supplied with the Sumoylation kit.