A Systematic Analysis of Host Factors Reveals a Med23-Interferon-? Regulatory Axis against Herpes Simplex Virus Type 1 Replication

Griffiths, Samantha J.; Koegl, Manfred; Boutell, Chris; Zenner, Helen L.; Crump, Colin M.; Pica, Francesca; Gonzalez, Orland; Friedel, Caroline C.; Barry, Gerald; Martin, Kim; Craigon, Marie H.; Chen, Rui; Kaza, Lakshmi N.; Fossum, Even; Fazakerley, John K.; Efstathiou, Stacey; Volpi, Antonio; Zimmer, Ralf; Ghazal, Peter; Haas, Jürgen

a) Co-immunoprecipitation. Co-immunoprecipitation was performed using the epitope-tagged plasmids pGBKT7 (Myc epitope) and pGADT7 (HA epitope) containing the T7 promoter, and recombinant vaccinia virus vTF-7 expressing the T7 RNA polymerase (NIH AIDS repository). 5610 6 HEK293 cells were seeded in 10 cm dishes and the following day infected with vTF-7 (MOI 10) for 1 h before transfecting 10 mg each of empty bait (pGBKT7) or Med23-Bait, and IRF-Prey (pGADT7) vectors with Lipofectamine (Invitrogen). After 48 h cells were lysed on ice for 30 min in NP40 buffer, containing protease and phosphatase inhibitors. Debris was removed by centrifugation, and protein quantified with a BCA protein assay kit (Thermo scientific, UK) as per manufacturers' instructions. Equal protein quantities (650 mg per sample) were pre-cleared by continuous mixing with 50 ml preequilibrated Protein G Sepharose beads for 1 h at 4uC. Samples were centrifuged and supernatants halved before overnight mixing incubation with beads pre-coated with 1 mg a-HA (Roche, UK) or a-c-Myc (Santa Cruz, UK) at 4uC. Beads with precipitated proteins were washed three times with ice-cold NP40 buffer, resuspended in 26 SDS protein sample buffer and boiled for 10 min before SDS-PAGE separation on two 8% polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes overnight at 4uC before blocking with 5% milk in TBS-Tween then incubating with either a-HA or a-myc antibody (diluted 1:1000 in 5% milk/TBS-Tween). Membranes were washed in TBS-Tween before incubation with HRP-conjugated secondary antibody (diluted 1:3000 in 5% milk/TBS-Tween). After further washes, proteins were detected by incubation with ECL Western blotting detection system, exposing to X-ray film and developing films in an OPTIMAX X-Ray film processor.