Peroxidase coupled secondary antibodies

Immunoblotting was performed according to standard procedures. Briefly, cells were washed in PBS and lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% [vol/vol] IGEPAL [Sigma], 1% [vol/vol] Na-deoxycholate, 0.1% [vol/vol] SDS, 1 mM DTT, 0.2 mM PMSF, 1 μg/ml leupeptin and pepstatin, 50 mM NaF, 0.1 mM Na-vanadate, and Complete protease inhibitor [Roche]). Equal amounts of protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Immunoblot analysis was performed using mouse monoclonal antibody (MAb) anti-β-actin (Sigma), mouse MAb recognizing HCMV pUL83/pp65 (3A12; Abcam), and rabbit polyclonal anti-IκBα (sc-371; Santa Cruz). The proteins were visualized using peroxidase-coupled secondary antibodies (Dianova) and the ECL chemiluminescence system (Cell Signaling).

Immunoblotting was performed as described previously (26 (link)) using antibodies recognizing the SARS-CoV-2 N protein (ABIN6952435) or GAPDH (sc-25778, Santa Cruz). Lysates were inactivated by sequential heat incubation (10 min at 70°C and 10 min at 95°C) before they were discharged from the BSL3 laboratory. Proteins were visualized using peroxidase-coupled secondary antibodies (Dianova) and the ECL chemiluminescence system (Cell Signaling Technology).