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

The in vitro thiolester reactions were performed based on the ISG15 conjugation initiation kit (Boston Biochem). All samples were prepared in 20 mL containing 50 mM HEPES pH 8.0 and 100 mM NaCl. The concentration of the recombinant proteins is indicated in the Figure Legend Figure S1 The selected stable 2fTGH cell clones express comparable levels of the TAP-tagged ISG15 orthologues. 2fTGH cells were stably transfected with the indicated TAP-tagged constructs as described in Materials and Methods. Four clones were selected for the TAP experiments, 2 clones expressing TAPtagged HuISG15 and 2 clones expressing TAP-tagged Ag-mISG15. One cell-line stably expressing TAP-tagged PRMT (Protein Arginine N-Methyltransferase) was used as a control (lane 2). The different cell-lines were seeded at the same density in 12well plates. 16 h after seeding, the cell-lines were stimulated with IFNb(1 ng/ml). One non-stimulated (ns) control was included (lane 1). 26 h after IFNbtreatment, cell lysates were prepared and separated by SDS PAGE. (A) Western blot using anti-FLAG Ab, revealing the TAP-tagged constructs. Open arrow indicates the ectopic expressed TAP-tagged ISG15 constructs. The PRMT control construct is a bigger protein. (B) Western blot using anti-HuISG15 Ab (gift of Dr. E Borden). Open arrow indicates the ectopic expressed TAP-tagged ISG15 constructs (note the weaker cross-species recognition of AgmISG15 by the antibody). Closed arrow indicates the induced endogenous ISG15 as a result of the IFN stimulation.