Pcmv6 an myc ddk tagged vector

JNK1α1 and JNK1α2 were amplified from pCDNA3 FLAG JNK1α1 (Addgene) and pCDNA3 FLAG JNK1α2 (Addgene), respectively. JNK1β1 and JNK1β2 were amplified from the cDNA derived from SV40-fibroblasts. The full-length WT isoforms and truncated mutants were inserted into pTRIP-SFFV (82 (link)) and the pCMV6-AN-Myc-DDK tagged vector (OriGene), respectively. TA cloning and exon trapping were performed with the pCR4-TOPO vector (Thermo Fisher Scientific) and the pET01 vector (MoBiTec GmbH), respectively, according to the manufacturer’s instructions. Control siRNA (D-001810-10) and MAPK8 siRNA (L-003514-00) were obtained from Dharmacon.

The human canonical OTULIN cDNA open reading frame clone was amplified from pCMV6-Entry FAM105B (OriGene) and inserted into the pCMV6-AN-Myc-DDK-tagged vector (OriGene) or pLentiIII-UBC (Abmgood). Site-directed mutagenesis to generate the OTULIN variants present in the patients was performed by a modified overlap-extension PCR-based method (95 (link)). All constructs were validated by Sanger sequencing. HEK293T cells were transiently transfected in the presence of Lipofectamine LTX (Thermo Fisher Scientific). For lentiviral transductions, semi-confluent HEK293T cells were cotransfected with OTULIN, GAG, POL, and ENV plasmids. The supernatant was collected at 48 hours and 72 hours and concentrated with a Lenti-X concentrator (Clontech). A ten-fold dilution (by volume) of the lentivirus preparation was added to semi-confluent immortalized fibroblasts plated on six-well plates. Transduced cells were selected with puromycin (Invitrogen). PDFs were mixed with gene knockout kit V2 single guide RNA pools (Synthego) in the presence of TrueCut Cas9 protein V2 (Thermo Fisher Scientific), or with pCMV6 expression vectors, and nucleofected with the basic nucleofector kit for primary mammalian fibroblasts (Lonza) on a Nucleofector 2b device (Lonza).