NP Gene Cloning and Co-IP Assay

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).