Plasmid and siRNA Transfection Protocols

The plasmids pCMV-HA-ISG15, pSG5-UBE1L, pCMV2-UBCH8, and pcDNA4-USP18 were described before [16 (link), 19 (link)]. Full-length PTEN cloned into a MYC-tagged expression vector [10 (link)] was obtained from Dr. Li Ma (MD Anderson Cancer Center). The pcDNA3-GFP-PTEN and the pCMV-GFP-USP18 plasmids were purchased (Addgene and GeneCopoeia). Deletions in PTEN were constructed (GENEWIZ). DNA sequence analysis confirmed the desired species were engineered. Respective vector controls were purchased. RISC-free control siRNA and two siRNAs independently targeting USP18 were purchased (GE Dharmacon). These sequences were: murine USP18 siRNA 1 (5′-CGTTGTTTGTCCAGCACGA-3′) and murine USP18 siRNA 2 (5′-AGGAACTCGAGGACGGAAA-3′). Plasmids and siRNAs were transfected into cells using Lipofectamine 2000 reagent (Invitrogen) and Opti-MEM medium (Gibco Thermo Scientific).