Irdye 800cw anti mouse igg

Vero cells were seeded in a 24-well plate (2 × 10⁵ cells/well) and incubated overnight to produce a confluent monolayer. The culture medium was removed the next day and the cells were washed once with 1 × PBS. Then, a 200 µL aliquot of ten-fold serially diluted DENV-2 or DENV DIP in DMEM supplemented with 10% (v/v) FBS was added to each well in triplicate and incubated for 2 h at 37 °C. The plates were rocked every 20 min and then 800 µl of 1% (W/V) high viscosity carboxymethyl cellulose overlay (Sigma Aldrich) in Medium 199 (Sigma Aldrich) containing 2% FBS was added. After 5 days of incubation, the cells were washed with PBS and fixed with cold (−20 °C) 1:1 (v/v) acetone/methanol for 5 min at room temperature. The fixative was removed and the cells were air dried for 1 h at room temperature. The cells were blocked with Odyssey blocking buffer (LI-COR Biotechnology) for 1 h at 37 °C and probed with mouse anti-Flavivirus E antibodies 4G2 (produced from culture supernatant of hybridoma D1-4G2-4-15) for 1 h at 37 °C, followed by detection with IRDye 800CW anti-mouse IgG (LI-COR Biotechnology) using LI-COR Odyssey CLx infrared imaging system (LI-COR Biotechnology).

Western blotting was performed as previously described.6 Briefly 10⁷ Jurkat T cells were lysed in 200 μl Triton X‐100 buffer and 2·5 × 10⁵ to 7·5 × 10⁵ cells were resolved by SDS–PAGE under reducing conditions. Protein bands were detected by the LI‐COR Odyssey Sa system after developing with rabbit anti‐SAP antibody (clone FL‐128 Santa Cruz Biotechnology (Santa Cruz, CA)), mouse anti‐phosphotyrosine (clone PT‐66, Sigma, St Louis, MO), goat anti‐HA (biotinylated, Vector Laboratories) or anti‐β‐actin (clone AC‐15, Santa Cruz Biotechnology) followed by the appropriate secondary antibody or fluorophore‐conjugated streptavidin (IRDye 680LT‐conjugated anti‐rabbit IgG, IRDye 800 CW anti‐mouse IgG, IRDye 680LT‐conjugated anti‐mouse IgG, IRDye 800 CW Streptavidin; all LI‐COR Biosciences, Lincoln, NE). Quantification of protein expression was performed using LI‐COR odyssey sa software version 1.0.

The HEK 293 S cells were plated one day before transfection in a 6-well plate. The pEG BacMam vectors containing target cDNAs were transfected with Lipofectamine 2000 (Invitrogen/Thermo Fisher) and incubated for 2 days. Cells on the plate were washed in PBS before scraping. Cell membranes were solubilized in lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol) plus 0.2% Triton X-100 and Protease Inhibitor Cocktail (Roche) for 1 h at 4 °C. Cell debris were pelleted by centrifugation. The supernatants of cell lysate were incubated with either pre-equilibrated GFP nanobody-conjugated NHS-Activated Sepharose 4 Fast Flow Agarose (GE Healthcare) or Pierce Protein A Agarose (Invitrogen/Thermo Fisher) with rabbit anti-Myc antibodies for 30 min at 4 °C. The resins were extensively washed in lysis buffer plus 0.1% Triton X-100 within 5 min at 4 °C. In all, 4× Laemmli Sample Buffer (Bio-Rad) was added, and samples were run in SDS-PAGE without extra elution steps. Bands of target proteins were visualized by western blotting (Supplementary Fig. 7) with mouse anti-GFP (Invitrogen/Thermo Fisher) (1:1000), anti-Myc (1:500), and anti-HA (1:500) antibodies as primary antibodies and IRDye-800CW anti-mouse IgG (Licor) (1:5000) as the secondary antibody. Images were taken in an Odyssey infrared scanner (Licor).