Goat anti mouse hrp conjugated second antibody

293T cells were seeded at 2 × 10⁵ cells/well in 6-well plate were transiently transfected with 4 µg of expression vectors along with the empty vector control using Lipofectamine 2000 (Invitrogen). After 2 days, the transfected cells were lysed in NP-40 lysis buffer and the cell lysates were analyzed on an immunoblot probed with mouse anti-FLAG antibody (Sigma-Aldrich) and anti-GAPDH antibody (Life Technologies) followed by goat anti-mouse HRP-conjugated second antibody (Sigma-Aldrich).

CHME3 cells (2 × 10⁵) were transfected with RTP4 expression vectors using Lipofectamine 2000. One dpi, the cells were infected with HCoV-OC43 at an MOI of 0.5 and 5 and incubated at 33°C. The cells were lysed on days 1 and 3 and the lysates were analyzed on an immunoblot probed with mouse anti-coronavirus OC43-N antibody (Sigma-Aldrich) followed by goat anti-mouse HRP-conjugated second antibody (Sigma-Aldrich). The infectivity for experiments was quantified by qRT-PCR. All HCoV-OC43 infections were done at Biosafety Level 2 (BSL2). SARS-CoV-2 (MOI = 0.1), Omicron BA.1, and BA.2 (MOI = 0.05, 0.5) infections in a BSL3 facility according to institutional guidelines provided by the NYU Langone and Institutional Animal Care and Use Committee according to the standards set by the Animal Welfare Act.

CHME3 cells were transiently transfected with hRTP4-2F expression vector by lipofection with Lipofectamine 2000 (Invitrogen) and then infected with HCoV-OC43 at an MOI of 5. After 2 days, the transfected cells were lysed in NP-40 lysis buffer (10 mM Tris HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, and protease inhibitor cocktail). The cell lysates (10 µg) were incubated for 1 h at 4°C with 20 µL anti-FLAG M2 magnetic beads (Sigma-Aldrich). The beads were pulled down in a magnetic separator and washed with buffer containing 50 mM Tris HCl, pH 7.5, and 150 mM NaCl. Bound protein complexes were eluted in buffer containing 0.1 M Glycine HCl, pH 3.0. The eluted proteins were analyzed on an immunoblot probed with mouse anti-FLAG antibody (Sigma-Aldrich) followed by goat anti-mouse HRP-conjugated second antibody (Sigma-Aldrich). RNA that had been pulled down with the RTP4 was isolated by phenol extraction followed by ethanol precipitation. Genomic and subgenomic HCoV-OC43 RNAs that have been pulled down were quantified by qRT-PCR.