Phoenix eco cells

The CAD-MigR1 retroviral construct was prepared by digesting 2 μg MigR1 plasmid with EcoRI and XhoI (NEB) at 37 degrees for one hour. HA-FLAG-CAD (provided by Dr. Brendan Manning, Harvard University) was digested with amplified with primers to introduce recognition sites for the EcoRI and XhoI cuts made in MigR1. The resulting product was transformed into DH5α (ThermoFisher), allowed to grow overnight at 37 °C, then isolated using a QIAprep Spin Miniprep Kit (Qiagen, 27104). The isolated products were then sequenced to ensure proper addition of the CAD gene sequence to the MigR1 backbone. For isolation of virus, Phoenix-ECO cells (ATCC, CRL#3214) were plated on 6 well plates at 1.2×10⁶ cells per well and allowed to grow in antibiotic-free DMEM for 24 hours before exposure to plasmid and Lipofectamine® 3000 per protocol (ThermoFisher). Media was changed to DMEM with antibiotics 24 hours after transfection with resulting supernatant harvested and filtered through a 0.45-micron filter and incubated with Retro-X® Concentrator (Takara, 631456) for viral isolation. The viral products were then introduced to T cells 24 hours post-activation (see Cell Cultures section) by spinfection at 2500 rpm for 2 hours at 37 °C in the presence of 5 μg/ml polybrene. Media was changed to remove polybrene 24 hours after spinfection.

The mouse Ta3 full-length cDNA was created by combining three cDNA fragments that were amplified from a cDNA library by PCR. Murine retroviral pLNCX-FH-Ta3 or its mutant cDNA constructs (numbers are amino acid positions) were created by inserting the cDNA fragments into pLNCX-FH retroviral vector by PCR and general cloning techniques. pLNCX-FH was engineered by inserting FH, FLAG and HA double tags, into pLNCX (Clontech). pRK-Ta3 and its mutant cDNA constructs were generated by inserting the cDNA fragments into a CMV based pRK or pRK-myc vector using PCR and general molecular cloning techniques. PKARIα cDNA was amplified from a mouse cDNA library by PCR and inserted into the pCMV-FLAG (Sigma) vector to create pCMV-FLAG-PKARIα. The constructs were verified by DNA sequencing. Virus carrying FH-Ta3 or its mutant constructs was generated by cotransfecting Phoenix-Eco cells (ATCC) with each of the viral constructs and pEco packaging construct using the calcium phosphate precipitation method (Wang et al., 2013 (link)). pRK-Gli2, pRK-Gli3, pRK-Gli2-P2, pRK-Gli3-P2 were described previously (Pan et al., 2006 (link); Wang et al., 2000 (link)).

Replication-incompetent retroviruses were generated by co-transfection of Phoenix-ECO cells (ATCC CRL-3214) with each MSCV-based retroviral plasmid and the pCL-Eco packaging plasmid using Lipofectamine 2000 (Thermo Fisher Scientific). A T cell hybridoma line lacking TCR alpha and beta chain gene expression (5KC_73.8.20) (White et al., 1993 (link)) or a murine B cell line (M12C3) (Glimcher et al., 1985 (link)) were spin-infected with retroviruses carrying each gene of interest as described previously (Nakayama et al., 2012 (link); Bettini et al., 2013 (link)). Transduced cells were selected by treatment with G418 sulfate (Thermo Fisher Scientific, 750 μg/ml for 5 days) and/or puromycin (Thermo Fisher Scientific, 3 μg/ml for 3 days) or flow cytometric cell sorting of fluorescence-positive cells according to the selection genes carried by the retroviral plasmids.

Flag-tagged Dvl2, unphosphorylatable β-catenin, HA-ubiquitin and NICD-myc plasmids were obtained from Addgene. Retroviral pLEX expression system was from Thermoscientific. Epsin 1 and epsin 1 ΔUIM plasmids and lentiviral pLL3.7 RNA interference vectors encoding epsins 1 and 2 shRNA were described previously^{29 (link)}. All remaining truncation and single domain constructs were generated using standard subcloning procedures. Epsin 1 point mutations were made using QuikChange Site-Directed Mutagenesis Kit per manufacturer’s instruction. Epsin 1 point mutations were predicted using three-dimensional modeling by PEP-FOLD (http://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD). Cloning and mutagenesis primers are available upon request. HEK 293T cells were transfected using Lipofectamine 2000 as instructed by the manufacturer. Lentiviral production was accomplished by transfecting Phoenix ECO cells (ATCC#CRL-3214) using Lipofectamine 2000. Viruses were harvested 48 hours post-transfection.