Mouse anti gfp mab

AP-61 cells were transduced with the recombinant baculovirus at an MOI of 20. After incubation for 24 h, the cells were washed with PBS (Gibco), the culture medium was refreshed, and incubation was continued for another 2 days. Vero cells were infected with CHIKV VRPs at an MOI of 0.05 prior to incubation in MEM at 28°C for 1 h. The cells were then washed using PBS, the culture medium was refreshed, and incubation continued for another 20 h at 28°C. The cells were washed with PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific). Protein samples were separated on a 4% to 12% Bis-Tris gel (Invitrogen). Western blot analysis was conducted following the transfer of proteins onto a nitrocellulose membrane using primary antibodies of either rabbit anti-CHIKV nsp1 polyclonal antibody (GeneTex) (1:5,000), mouse anti-CHIKV capsid MAb (Native Antigen Company Ltd.) (1:1,000), anti-CHIKV E2 MAb (1:1.000) (1b1 from Taiwan CDC), rabbit anti-E1 serum (45 (link)), mouse anti-GFP MAb (Santa Cruz Biotechnology, Inc.) (1:1,000), or mouse anti-actin MAb (Sigma-Aldrich) (1:5,000), as well as secondary antibodies of either goat anti-mouse or rabbit secondary antibody conjugated to horseradish peroxidase (Sigma-Aldrich). After incubating the membranes with an ECL substrate, signals were captured using the Amersham Imager 600 (GE Healthcare).

Mouse anti-Lamp1 mAb (H4A3; 1:500 for immunofluorescence or hereafter IF), mouse anti-CD63 mAb (H5C6; 1:100 for IF), and mouse anti-CD8a mAb (OKT8; 1:500 for IF) were from the Developmental Studies Hybridoma Bank. Mouse anti-GFP mAb (catalog no. sc-9996; 1:1000 for Western blotting) was purchased from Santa Cruz Biotechnology, Inc. Mouse anti-EEA1 mAb (catalog no. 610456; 1:500 for IF) and mouse anti-GM130 mAb (catalog no. 610823; 1:500 for IF) were from BD Biosciences. Horseradish peroxidase–conjugated goat anti-mouse (catalog no. 176516; 1:10,000 for Western blotting) was from Bio-Rad. Alexa Fluor–conjugated goat antibodies against mouse IgG (1:500 for IF) were from Thermo Fisher Scientific.
PNGase F was purchased from New England Biolabs. The following small molecules were commercially available: nocodazole (Merck; working concentration: 33 μm), brefeldin A (Epicenter Technologies; working concentration: 10 µg/ml), cycloheximide (Sigma–Aldrich; working concentration: 10 µg/ml), and GalNAc-O-Bn (Sigma–Aldrich; working concentration: 2 mm).