The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection

Galindo, I; Hernáez, B; Muñoz-Moreno, R; Cuesta-Geijo, M A; Dalmau-Mena, I; Alonso, C

The Vero-adapted ASFV isolate BA71V was used. For flow cytometry (FACS) analyses we used an infectious recombinant ASFV, B54GFP-2, which expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the EGFP. 41 Preparation of viral stocks, titrations and infection experiments were carried out in Vero cells as previously described. 42 The plasmid pCMVshort-EGFP-ATF6a was kindly provided by Dr. Mori (Kyoto University, Japan) 32 and was transfected into Vero cells using Fugene 6 (Roche Biochemicals, Mannheim, Germany), following the manufacturer's protocols. Western blotting. Cells seeded in 24-well plates were harvested in Laemmli sample buffer, boiled for 5 min at 95 1C, resolved by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The non-specific antibody binding sites were blocked with skimmed milk in phosphate buffered saline (PBS) and then incubated with the specific primary and the HRP (Horseradish peroxidase)-conjugated secondary antibody.