Fig. 6.

The identification of epitope recognized by SeP-neutralizing Abs. a Schematic outline of the epitope mapping approach. Green fluorescence protein (GFP)-tagged deletion constructs of hSeP were prepared. b Identification of AE2 mAb recognition site. Whole-cell lysates transfected GFP-tagged deletion constructs of hSeP were analysed by western blotting using anti-hSeP Ab AE2 and anti-GFP Ab. c Amino acid sequence homology around the first histidine-rich region (FHR) between three species. Predicted epitope of neutralizing antibody is enclosed. d Specificity and cross-reactivity of prepared polyclonal antibody for the FHR of mouse SeP (mFHR). Serum from wild type and SeP-KO mouse and other animal species was analysed by western blotting using mFHR pAb. As a loading control, separated proteins were stained with Coomassie Brilliant Blue R-250 (CBB). The major band derived from albumin is indicated. e Immunoprecipitation of mSeP by mFHR pAb. Mouse SeP in mouse serum was reacted with mFHR pAb- or control IgG-conjugated beads 4 °C for 2 h, and then immunoprecipitants were analysed by western blotting using mFHR pAb. f Evaluation of mFHR pAb against Se-supply activity by mSeP in C2C12 myocytes. Cells were treated with mouse serum (1%) in the presence of the indicated amount of mFHR pAb or control rabbit IgG (10 µg/mL) for 24 h. GPx1 levels were determined by western blotting