Fig. 5

Extracellular TfR1 interacts with TGEV S1 protein in vitro. a and b 293 T cells were co-transfected with a HA-tagged TGEV S1 expression plasmid together with plasmids expressing TfR1 or GFP-tagged TfR1-Out. Cell lysates were immunoprecipitated with anti-HA antibody and anti-TfR1 antibody, or anti-HA antibody and anti-GFP antibody, respectively. The precipitates were examined by western blotting using anti-HA antibody and anti-TfR1 antibody (a) or anti-HA antibody and anti-GFP antibody (b) to examine the interaction between HA-TGEV-S1 and TfR1, or HA-TGEV-S1 and GFP-tagged TfR1-Out, respectively. c His-tagged TfR1-Out was expressed in E.coli BL21 and purified using a Ni-NTA column. Purified products were separated using SDS-PAGE and stained with Coomassie brilliant blue. d Purified TfR1-Out was verified by western blotting. e and f IPEC-J2 cells infected with TGEV (MOI 5) were pre-incubated with TfR1-Out (200 ng/mL) for 1 h at 37 °C, and cell lysates were harvested for western blotting. The ratio of TGEV-N to GAPDH was normalized to control conditions, and culture supernatants were collected for viral plaque assays in ST cells. Plaques developed 2 days after infection. Normal cells and cells treated with pET-32a-c(+) served as controls. Data shown are means ± SD from three independent experiments. (* 0.01 < p < 0.05, ** p < 0.01)