We had previously detected NS1 as early as 5C6 hpi . study, 124 of which have not been previously reported. RNAi screens identified 11 NS1-interacting host factors as vital for IAV replication. Knocking down one of these, nuclear mitotic apparatus protein 1 (NUMA1), dramatically reduced IAV replication. IAV genomic transcription and translation were not inhibited but transport of viral structural proteins to the cell membrane was hindered during maturation steps in NUMA1 knockdown (KD) cells. for 2 h at 4 C. 2.2. Virus Titration Serial 1:10 dilutions of viral stocks and experimental samples were titrated by plaque assay in MDCK cells, using a 1:1 mixture of 1.2% type 1 agarose and 2 DMEM, supplemented with 2.5 g/mL LG 100268 Tosyl-L-lysyl-chloromethane hydrochloride (TLCK)-treated trypsin, as described . Plates were incubated at 35 C for 66 h, fixed with 2% formaldehyde and stained with crystal violet to determine viral plaque forming units (PFU) per mL. 2.3. Cytoplasmic and Nuclear Fractionation A549 cells were infected with PR8 at a MOI of 5 PFU/cell. Mock-infected controls were treated similarly but without virus. Cells were harvested and processed as described  with minor modifications. Briefly, infected and mock-infected cells were scraped from plates at 6 and 24 h post infection (hpi), washed 3 with ice-cold phosphate buffered saline (PBS), cellular pellets resuspended in lysis buffer (150 mM NaCl, 10 mM Tris, pH 7.5, supplemented with 0.4% NP40 and 1 Roche complete?-ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor, Mississauga, ON, Canada) on ice for 15 mins and vortexed every 5 min. Cytoplasmic extracts were prepared by centrifuging for 5 min at 500 for 10 min. The protein concentrations of all cytoplasmic and nuclear extracts were determined by a Pierce? bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA). 2.4. Co-Immunoprecipitation (Co-IP) Cytoplasmic and nuclear lysates were initially pre-cleared with non-coupled protein Rabbit Polyclonal to AKT1/2/3 (phospho-Tyr315/316/312) G Dynabeads (Invitrogen, Waltham, MA, USA) for 90 min at 4 C. The pre-cleared lysates were clarified at 10,000 for 7 min. Dynabeads were washed 3 with TBST (Tris-buffered saline supplemented with 0.05% Tween 20) and a mixture of anti-NS1 mAbs 3F5, 5F4 and 4E10, which recognize different NS1 epitopes , was added to the beads. The mAbs and beads were incubated at room temperature for 90 min in a rotator to allow Ab-bead binding. Monoclonal -Emprin (IgG2a), monoclonal -SYN (IgG2b) and monoclonal -HSA (IgG1) antibodies (gift from Dr. Wilkins, Manitoba Centre for Proteomics and Systems Biology) also were bound to Dynabeads to serve as isotype controls. Ab-coupled beads were washed 4 with TBST to remove unbound mAbs and mixed with the pre-cleared cellular fractions in a rotator overnight at 4 C. The unbound fractions were discarded and beads were washed 4 and resuspended in TBST. The washed and resuspended bead-Ab-antigen complex represented immunoprecipitated (IP) products. Co-IPs were also performed after coupling anti-NUMA1 (Bethyl Laboratory, A301-510A), anti-PRPF19 (Bethyl Laboratory, A300-101A) and anti-UTP6 (Thermo Fisher, PA5-21716) antibodies to Dynabeads. 2.5. Processing of IP Product for Western Blot Analysis and Mass Spectrometry The IP products and beads were washed 2 with RIPA buffer, 1 with ammonium bicarbonate buffer supplemented with 0.1% NP40 and resuspended in ammonium bicarbonate buffer. 10% of the resuspended bead mixtures were dissolved in sodium dodecyl sulfate (SDS) running buffer and resolved in 4C12% gradient Novex NuPAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Gels (Invitrogen, Waltham, MA, USA) for Western blot analysis and 90% of the resuspended beads were saved at ?80 C for subsequent mass spectrometry (MS) analysis. For MS analysis, the immunoprecipitated beads were digested overnight with 1 g of trypsin in 100 mM ammonium bicarbonate solution at 37 C. After tryptic digestion, equal volumes of trifluoroacetic acid (TFA)/Acetonitrile (ACN) (100% ACN & 1% TFA) were added to the digested IP products and vortexed 10C15 min. Digested peptides were separated from beads by centrifugation at 17,000 for 5 min and were dried in a Savant SpeedVac vacuum dryer. Dried peptides were LG 100268 resuspended in 50 L of 0.5% TFA and desalted with C18 ziptips. Eluted peptides LG 100268 were analyzed in an AB SCIEX (Concord, ON, Canada) Triple TOF 5600 mass.