2.10. Pull-Down Assay

CIGB-325-interacting proteins were identified by in vitro pull-down followed by tandem mass spectrometric analysis (LC-MS/MS). MDBK cells were seeded at 3,600,000 cells in 60 mm dishes and infected with 500 µL of virus at 70,000 TCID₅₀/dish (MOI = 0.01). After 48 h post-infection, cells were collected, washed, and lysed in hypotonic PBS solution (1×) (Sigma, St. Louis, MO, USA) containing 1 mM DTT (Sigma, St. Louis, MO, USA), TritonX-100 (1%), and complete protease inhibitor (Roche, Basel, Switzerland). Cellular lysates were cleared by centrifugation, and 300 µg of total protein was incubated with biotin-tagged CIGB-325 (100 μM) or medium alone for 30 min at room temperature and added to 30 µL of pre-equilibrated streptavidin–sepharose matrix (GE Healthcare, Chicago, IL, USA). Following 1 h at 4 °C, the matrix was collected by centrifugation and extensively washed with cold PBS 1 mM with DTT. CIGB-325-interacting proteins were eluted, resolved in an SDS-PAGE gel, and processed as described later. For in vivo pull-down assays, BCoV-Mebus-infected and uninfected MDBK cells were treated with biotin-tagged CIGB-325 (100 μM) or medium alone for 30 min at 37 °C in 5% CO₂. Subsequently, cells were collected and pull-down assay was conducted as mentioned above. Proteins bound to streptavidin–sepharose were eluted, resolved in a 12 % SDS-PAGE gel, and transferred onto nitrocellulose membranes. For Western blot analysis, a human polyclonal IgG anti-SARS-CoV-2 and a mouse monoclonal antibody against CK2α (Abcam, Cambridge, UK) were used as primary antibodies. Detection was performed with peroxidase-conjugated anti-mouse IgG 1:5000 (Sigma, St. Louis, MO, USA) and anti-human IgG 1:100 (Jackson ImmunoResearch, West Grove, PA, USA). In parallel, untreated cells were subjected to the same experimental procedure to identify those proteins non-specifically bound to streptavidin–sepharose matrix.