2.9. HBEGF and MUC5AC Protein Expression Verification

The level of HBEGF and MUC5AC protein was measured using an HBEGF ELISA kit (Assay Genie, Dublin, Ireland) and MUC5AC ELISA assay kit (Cusabio Biotech, Houston, TX, USA) following the manufacturer’s protocol. HT-29 cells were stimulated by CotG-p75 (10⁷ spores/mL) for 0, 3, 6, and 12 h. The cells treated with no spores or wild-type spores were used as controls. The amount of HBEGF and MUC5AC protein in cell lysates and culture supernatants was normalized to total protein in cell lysates and culture supernatants. The amount of total protein was determined using the BCA protein assay (Pierce, Thermo Scientific, Waltham, MA, USA). The results were expressed as relative expression by normalizing protein expression levels for CotG-p75-treated conditions versus control at all time courses.