Molecular Tools for ISG15 Signaling

Plasmids pCMV-HA-ISG15, pSG5-UBE1L, pCMV2-UBCH8, pcDNA4-USP18, pCGN-KRAS(G12V), pPUR and pRetroX-IRES-ZsGreen1-USP18 were described before (6 (link),8 (link),18 (link)). Lentiviral pCMV-dR8.2 dvpr and pMD2.G plasmids were purchased (Addgene) along with the pCMV-GFP-USP18 plasmid (GeneCopoeia). Candidate TRC pLKO.1 lentiviral shRNAs repressing USP18 were purchased from GE Dharmacon. Respective vector controls were purchased. QuikChange II Site-Directed Mutagenesis Kits (Agilent) rendered pcDNA4-USP18 enzymatically-inactive, as described (8 (link)). DNA sequence analysis confirmed desired engineered species. RISC-free control siRNA and two siRNAs independently targeting USP18 were purchased (GE Dharmacon). These sequences were: murine USP18 siRNA 1 (5′-CGTTGTTTGTCCAGCACGA-3′), murine siRNA 2 (5′-AGGAACTCGAGGACGGAAA-3′), human USP18 siRNA 1 (5′-CTGCATATCTTCTGGTTTA-3′), and human USP18 siRNA 2 (5′GGAAGAAGACAGCAACATG-3′). Plasmids and siRNAs were transfected into desired cells using Lipofectamine 2000 reagent (Invitrogen) and Opti-MEM medium (Gibco Thermo Scientific).