Expression of CHIKV E1 proteins in Sf21 cells infected by recombinant baculoviruses. (A) Sf21 insect cells were grown and infected with recombinant viruses at an M.O.I. of one. Total protein was harvested at 2 dpi and separated on 10% SDS-PAGE. Viral glycoproteins were detected by Western blot using rabbit anti-CHIKV E1 serum (upper gel), then re-probed with anti-baculovirus gp64 antibody (lower gel). Two protein size markers are indicated on the left. (B) Detection of E1 on the cell surface by biotinylation assay. Baculoviruses infected-Sf21 cells as indicated were labeled with biotin and lysed. Biotinylated surface proteins were resolved by 10% SDS-PAGE and detected by Western blot using rabbit anti-CHIKV E1 serum (upper blot), and then re-probed with anti-baculovirus gp64 antibody (lower blot). Cell conditions and protein markers are given by the legend of (A). (C) Immunofluorescence analysis of CHIKV E1 on the cell surface. Sf21 cells infected with the indicated recombinant baculoviruses were stained with anti-CHIKV E1 antibodies followed by secondary goat anti-rabbit IgG antibodies labeled with Alexa Fluor 546. Cells were examined and photographed using a fluorescent microscope under identical green and red lighting conditions. Overlaid images show green fluorescence representing the infected-Sf21 cells expressing EGFP, and red fluorescence representing CHIKV E1 protein signals (indicated by arrows). Cells without EGFP, but stained in red were dead cells. The bar represents 10 μm.