Pa522819

Antibodies used in this study include rabbit anti-Syncytin-1 (for western blot, PA522819; Invitrogen), rabbit anti-Syncytin-1 (for flow cytometry, PA522819; Invitrogen), mouse anti-bZIP (sc69797; Santa Cruz), mouse anti-K8.1 (sc65446; Santa Cruz), rabbit anti-KSHV RTA (a gift from Yoshihiro Izumiya at University of California Davis), mouse anti-β-actin (AC-15; Sigma), rabbit anti-GAPDH (2118S; Cell Signaling), mouse anti-EA-D (MAB8186; EMD), mouse IgG1 negative isotype (CBL610; EMD Millipore), mouse anti-ZEBRA (a gift from Paul Farrell at Imperial College London, London, United Kingdom), rabbit anti-FLAG (F7425; Millipore), rat anti-EBNA2 (MABE8; Millipore), mouse anti-BrdU (555627; Thermo Fisher Scientific), mouse anti-CD19-APC (MABF197; Sigma-Aldrich), mouse anti-CD20-PE (MABF1635; Sigma-Aldrich), mouse IgG1-APC isotype (550824; BD Pharmingen), rat IgG2a isotype (MABF1077Z; EMD Millipore), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) (AP308P; EMD Millipore), HRP-conjugated goat anti-rabbit IgG (AP307P; EMD Millipore), FITC-conjugated goat anti-mouse IgG (F0257; Sigma), and APC-conjugated goat anti-rabbit IgG. All antibodies were used at concentrations and conditions recommended by manufacturers.

A549 cells were transduced with the multiplicity of infection (MOI) of 10 for the relevant hAd19a/64 vaccine. Twenty-four hours later, cells were incubated with 15 µg/mL of primary rabbit anti-human HERV polyclonal antibody (PA5-22819; Invitrogen™, Waltham, MA, USA) in FACS buffer (PBS containing 1% BSA and 0.1% NaN₃) for 1 h at 4 °C. This antibody targets the HERV-W Env SU region. Next, cells were stained with secondary phycoerythrin (PE) donkey anti-rabbit IgG antibody (406421; BioLegend^®, San Diego, CA, USA; 1:100) and eBioscience™ Fixable Viability Dye eFlour™ 780 (65-0865; Invitrogen™ Waltham, MA, USA; 1:1000) for 30 min at 4 °C. Finally, cells were fixated in 1% paraformaldehyde (PFA) for 15 min at 4 °C. Flow cytometry was run on the LSRFortessa™ 3-laser cell analyser (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analysed using FlowJo™ v10 analysis software and GraphPad Prism 9.