Anti np antibody

The NP gene of H5N1 A/Hatay/2004 isolate was cloned into pCDNA 3.1-His plasmid to be used as bait vector for co-IP studies. Full-length human API5 gene cloned in HA and Flag-tagged pLPC plasmid was provided by Nicholas J Dyson.^{31 (link)} Anti-NP antibody was obtained from Abcam (Cambridge, MA, USA). Anti-API5, anti-APAF1, anti-procaspase 3, anti-cleaved caspase 3, anti-procaspase 9, anti-cleaved caspase 9, anti-myc, and anti-His antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Anti-Flag, anti-GAPDH, anti-API5, and anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-PARP antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Pool of gene-specific siRNAs against API5 was purchased from Santa Cruz Technologies (Santa Cruz, CA, USA).

MBCD and MTT (3-4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) were purchased from Sigma-Aldrich. For raft protein isolation, a focus signal proteins kit (G-biosciences, Saint Louis, MO, USA) was purchased and used according to the manufacturer’s protocol. To visualize lipid rafts, a Vybrant Lipid Raft Labeling Kit was purchased from Molecular Probes (Thermofisher Scientific, Waltham, MA, USA). For confocal microscopy and Flow Cytometric studies, Anti-NP-FITC (anti-nucleoprotein-fluorescein isothiocyanate) conjugated antibody was purchased from Abcam (Bristol, UK). For Western blotting, anti-GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-actin-HRP antibody (Sigma Aldrich, St. Louis, MO, USA), and anti-NP antibody (Abcam, UK) was used.

Lungs were collected to detect the relative expression of SIV NP protein. In brief, total proteins were extracted, and then the protein concentration was measured using a BCA kit (Beyotime, China). Subsequently, the proteins were denatured, subjected to SDS-PAGE, and then transferred to the PVDF membranes. Next, the membranes were blocked with 5% BSA, followed by overnight incubation at 4°C in anti-NP antibody (Abcam, United Kingdom) and 2 h of incubation with an anti-mouse secondary antibody (Cell Signaling Technology, United States) at RT. Finally, the bound antibodies were visualized using an enhanced chemiluminescence kit (Beyotime, China).