Figure 3
Recombined gI MW and deglycosylation analysis. (A) DEFs were transfected with gI expression plasmids (pCAGGS-gI-Flag, pCAGGS-gIN69A-Flag, pCAGGS-gIN78A-Flag, pCAGGS-gIN265A-Flag, and pCAGGS-gIN69/78/265A-Flag) at a dose of 1000 ng per well. Western blot analysis was performed on cell lysates 36 h post-transfection, and recombinant gI proteins were detected using a mouse monoclonal anti-Flag antibody: gI-Flag (lane 1), gIN69A-Flag (lane 2), gIN78A-Flag (lane 3), gIN265A-Flag (lane 4), and gIN69/78/265A-Flag (lane 5). (B) Collect the cell lysates from DEFs transfected with recombinant gI plasmids, and treat them with PNGase F at 37 °C for 1 h. The digested products of PNGase F are then subjected to 12% SDS-PAGE, followed by western blot. Recombinant gI is analysed using a mouse monoclonal anti-Flag antibody, with GAPDH serving as a loading control. Similarly, cell lysates from DEFs infected with recombinant viruses were processed in the same manner. Analysis of recombinant gI using a mouse polyclonal anti-gI antibody, with a rabbit polyclonal anti-VP5 antibody serving as a control for viral infection, and GAPDH as a loading control. The results presented are representative of three independent experiments.