HyperISGylation of Old World Monkey ISG15 in Human Cells

Pattyn, Els; Verhee, Annick; Uyttendaele, Isabel; Piessevaux, Julie; Timmerman, Evy; Gevaert, Kris; Vandekerckhove, Joël; Peelman, Frank; Tavernier, Jan

For standard ISGylation experiments in Hek239T cells, 3,5610 5 cells were seeded the day before transfection in 6-well plates and transfected for 6 h using a standard calcium phosphate precipitation procedure. Typically 0.75 mg of ISG15variant, if indicated supplemented with 0.25 mg E1 and 0.5 mg E2 or, applicable supplemented with 0.75 mg substrate was used in a total volume of 250 mL DNA/CaPO4-transfection mixture. FugeneHD (Roche) was used for transfection in COS cells and Lipofecta-mine2000 (Invitrogen) for transfection in N38 cells. Since FugeneHD induces a minimal IFN response, this reagent was exceptionally used for the Hek293T transfections in Figure 7b . However, no differences could be seen between this transfection reagent and the calcium phosphate precipitation method regarding the ISGylation difference between Hu and AgmISG15.