Mouse monoclonal α flag tag antibody

To investigate the expression of the novel vIL-8 splice variant and vIL-8 secretion, Western blot analyses were performed as described previously [20 (link)]. Briefly, CECs were infected with 10,000 plaque-forming units (pfu) of the indicated viruses. Cells or supernatants were harvested at 5 days post infection (dpi). Samples were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Carl Roth; Karlsruhe, Germany) using the Biometra semi-dry blotting system (Biometra; Göttingen, Germany). Subsequently, the membranes were blocked with 5% milk in phosphate-buffered saline (PBS) and incubated overnight at 4 °C with a mouse monoclonal α-FLAG tag antibody (1:1000; ABM; Richmond, Canada), the rabbit polyclonal anti-vIL-8 antibody [8 (link)], or the mouse monoclonal anti-gC antibody [9 (link),21 (link)], respectively. After three washes with PBST (PBS containing 0.05% Tween 20), the membranes were incubated for one hour at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit antibodies (1:10,000; Cell Signaling; Danvers, MA, USA). Finally, membranes were visualized using enhanced chemiluminescence (ECL) plus substrate (Thermo Fisher Scientific; Waltham, MA, USA), and protein signals were visualized with the Chemi-Smart 5100 detection system (Peqlab; Erlangen, Germany).

To investigate the protein expression of the novel splice form, indirect immunofluorescence analysis (IFA) was performed as described previously [20 (link)]. Briefly, CECs were infected with 100 pfu, fixed at 5 dpi with 4% paraformaldehyde, and blocked with 3% BSA for 30 min. Subsequently, cells were stained with the mouse monoclonal α-FLAG tag antibody (1:500; ABM; Richmond, Canada) and incubated for 45 min at room temperature. Cells were washed with PBS, probed with Alexa goat anti-mouse IgG (H + L) 568 antibody (1:1000; Invitrogen; Carlsbad, CA, USA), and incubated at room temperature for 45 min. After three washes with PBS, cells were stained with DAPI stain (5 μg/mL) in PBS. Cells were examined and images captured with an AxioVision microscope (Zeiss; Oberkochen, Germany).