Irdye 800cw goat anti rabbit igg secondary antibody

Cells were collected during mid-exponential growth and broken by vortexing with glass beads (150–212 µm diameter) in isolation buffer (25 mM MES/NaOH pH 6.5, 10 mM CaCl₂, 10 mM MgCl₂, 25% glycerol, and protease inhibitor, Roche). Vortexing was done in five consecutive cycles at 4°C, each cycle consisting of 1 min of vortexing followed by 1 min of rest. Unbroken cells and glass beads were removed by centrifugation (400 g, 1 min). Membrane and soluble fractions were then separated by centrifugation at 16,000 g for 20 min at 4°C. Protein concentrations were measured with the Pierce BCA protein assay kit (Thermo Scientific). In total, 10 µg protein per sample was used for SDS-PAGE separation. GFP was detected by western blotting using an Anti-GFP primary antibody (Abcam, 1:5,000 dilution in 10× PBS, 0.8% Tween 20, and 5% milk) followed by the IRDye 800CW Goat anti-Rabbit IgG secondary antibody (LI-COR, 1:5,000 dilution in 10× PBS, 0.8% Tween 20 and 5% milk).

For cell staining, cells were plated at 1 × 10⁴ cells/well in 96-well plates and incubated overnight. The cells were then infected with PeV-A3 at multiplicity of infection (MOI) = 5. After 8- and 24-h infection, the cells were fixed with 1:1 Methanol/Ethanol solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Cells were incubated with blocking buffer [10% skim milk in phosphate-buffered saline (PBS)] and incubated with anti-PeV VP0 polyclonal antibody at 4°C overnight (LTK BioLaboratories, Taoyuan, Taiwan) (10 (link), 29 (link)). Then, the IRDye^® 800CW goat anti-rabbit IgG secondary antibody was added into the cells (Li-COR, #926-32211, Lincoln, NE, USA), followed by 2-h incubation. Images of immunofluorescence assay were captured, and the fluorescence intensity of PeV-A3-infected cells were quantified using Odyssey image system (Li-COR, Lincoln, NE, USA).

Anti-DCP1, anti-EDC4 and anti-HA antibodies were purchased from Cell Signaling Technology (Danvers MA). Anti-DCP2 antibody was developed by Prestige Antibodies and purchased from Sigma-Aldrich (St. Louis MO). Anti-DDX6 antibody was developed by Thermofisher (PA5–18478). For immunoblotting, anti-EDC4 was purchased from Proteintech (Rosemont IL) and anti-EDC3 antibody was purchased from ABClonal (Woburn MA). Anti-βactin antibody was purchased from R&D Systems (MAB8929). Anti EBOV VP35 antibody was developed and obtained from Dr. Daisy Leung (Washington University, St. Louis MO). Alexa-Fluor 488, Alexa-Fluor 546 goat anti-mouse and goat anti-rabbit antibodies as well as Alexa-Fluor 488 chicken anti-goat, Alexa-Fluor 546 donkey anti-rabbit and Alexa-Fluor 637 donkey anti-mouse antibodies were purchased from Invitrogen (Thermofisher Scientific, Waltham MA). Chicken anti-BioID2 primary antibody was purchased from BioFront Technologies (BioBID-CP-100). IRDye 800CW donkey anti-chicken secondary antibody (LI-COR Biotechnology, 925–32218), IRDye^® 800CW Goat anti-Rabbit IgG Secondary Antibody (LI-COR Biotechnology, 925–32211, 1/10000), and IRDye^® 680RD Goat anti-Mouse IgG Secondary Antibody (LI-COR Biotechnology, 926–68070) were used as secondary antibodies for all relevant immunoblotting experiments.