Peroxidase conjugated goat anti rabbit antibody

SDS-PAGE was performed using standard techniques and was followed by a semidry blotting. Antibodies raised against MBP were prepared in rabbits [34] . The protein was visualized using secondary peroxidase-conjugated goat anti-rabbit antibodies (Sigma-Aldrich, St Louis, MO, USA).

Western blot analysis was performed to test the expression of MBP protein. Ten larvae were lysed in buffer with protease inhibitors (Complete Mini, Roche, Mannheim, Germany), and the protein concentrations were quantified with a BCA Protein Assay Kit (Complete Mini, Roche, Mannheim, Germany). The proteins were separated in a 10% SDS-PAGE gel and were transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was then blocked in 5% nonfat dry milk in PBS for 1 hour and incubated with anti-MBP (1∶500; Anaspec, Fermont, CA) and anti-GAPDH (1∶3,000; Millipore). The blots were rinsed with PBS and incubated with peroxidase-conjugated goat anti-rabbit antibodies (1∶5,000; Sigma-Aldrich, St. Louis, MO) for 1 hour. The bound antibody was visualized using an enhanced chemiluminescence assay (Millipore).

HEK293FTs and IPPK-KO cells were maintained, and plated for transfections and transductions, as previously described^{13 (link)}. RSV particles for cellular assays were produced and cells transduced in a similar manner as for HIV, as previously described^{13 (link)}. The flow cytometry gating strategy is shown in Supplementary Fig. 10. Western blots and their analysis were performed as previously described, and briefly here^{13 (link)}. A rabbit anti-RSV-capsid antibody (prepared in-house) was used at a 1:500 dilution in 5% nonfat dry milk in PBS-Tween20 for the 1 h primary application at room temperature. A goat anti-rabbit peroxidase-conjugated antibody (Sigma, A0545) was used at a 1:10,000 dilution in 5% nonfat dry milk in PBS-Tween20 for the 1 h secondary application at room temperature. Per the manufacturer’s protocols, membranes were stripped with Restore PLUS Western Blot Stripping Buffer (Thermo, 46430), re-blocked, incubated with a mouse anti-GAPDH antibody (Santa Cruz Biotechnology, SC-47724) at a 1:500 dilution, washed, incubated with goat anti-mouse-HRP (Sigma, A5278) at a 1:10,000 dilution, and imaged. Blot images were converted to 8-bit format, Fiji’s (ImageJ) gel analysis tools were used to calculate blot densities, and values were exported to a CSV file for subsequent analysis in RStudio.