Fig. 4.

Membrane expression of mutants at E267 of hPEPT1 in transiently transfected HEK293 cells after 72 h posttransfection. Membrane localization was visualized with affinity-purified rabbit anti-hPEPT1 primary antibody (1:200) and FITC-conjugated secondary antibody (1:200). HEK293 cells with transfected proteins were biotinylated with sulfo-NHS-LC biotin for 30 min at room temperature. Immunoprecipitation was carried out, followed by Western blot analysis using affinity-purified rabbit anti-hPEPT1 primary antibody (1:500), with visualization using goat anti-rabbit HRP-conjugated secondary antibody (1:10,000) and chemiluminescence. α₁-integrin (1:10,000) was used as the internal control