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

Immunofluorescence. Cells seeded on glass coverslips in a 24-well plate were infected with ASFV. At several time points after virus infection, the cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized in 0.1% Triton X 100 in PBS for 15 min at room temperature. Cells were consecutively stained with primary and secondary antibodies and then incubated with Topro-3 (Invitrogen, Eugene, OR, USA) 1/1000 in PBS1X for DNA staining. After washing, coverslips were finally mounted on glass plates and cells were observed under a Leica TCS SPE confocal microscope (Leica-Microsystems, Wetzlar, Germany). The primary antibodies used were: rabbit polyclonal antibody to GRP78 (BiP) (Santa Cruz Biotechnology) 1 : 50, rabbit polyclonal antibody to caspase 12 (BioVision) diluted 1 : 25 and anti-ASFV p30 monoclonal antibody 1 : 100. Antirabbit IgG Alexa 488 and anti-mouse IgG Alexa 594-conjugated antibodies (Invitrogen) diluted to 1 : 200 were used as secondary antibodies respectively.