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

Two days after transfection, cells were lysed with 150 mL 26 SDS loading buffer (62 mM Tris HCl pH 6.8, 2% SDS, 8% glycerol, 5% b-ME, 0.01% brome phenol blue) and homogenized on a Qiashredder mini spin column (Qiagen). 35 mL of the boiled lysate was loaded on a SDS-polyacrylamide gel. Blotting efficiency was checked using Ponceau S staining (Sigma). The precision plus protein standard all blue (Biorad) was used as molecular weight marker. FLAG-tagged proteins were revealed using a 1/8000 dilution of anti-FLAG M2 mouse monoclonal Ab (Sigma), V5-tagged proteins by 1/5000 dilution of anti-V5 mouse monoclonal Ab (Invitrogen). Hu and OWmISG15 could be detected by an anti-human ISG15 rabbit polyclonal Ab (Abcam). Human ISG15 was also detected with the anti-human ISG15 mouse monoclonal Ab, a generous gift from Dr. E. Borden. Anti-human actin rabbit polyclonal Ab (Sigma) 1/3000 diluted was applied as control loading. Either goat anti-mouse IRDyeH 800CW or anti-rabbit IRDyeH 680 (LI-CORH Biosciences) was used as secondary Ab. Targeted proteins on the blots were visualized using the OdysseyH infrared imaging system (LI-CORH Biosciences).