Far-Western Blot Analysis of rTsCTL Binding

The binding of rTsCTL and IEC was investigated by far-Western blot analysis as previously described [16 (link)]. In brief, soluble IEC proteins were first separated by SDS‒PAGE and then transferred to NC membranes (Millipore, USA). The membrane was cut into strips, blocked with 5% skim milk at 37 °C for 2 h, and then incubated with 20 μg/mL rTsCTL for 2 h at 37 °C. Following washes with PBST, strips were probed at 37 °C for 1 h with anti-rTsCTL serum (1:100) as the primary antibody and HRP-anti-mouse IgG (1:10 000; Southern Biotech) as the secondary antibody [52 (link)]. After washing, colouration was developed using 3-amino-9-ethylcarbazole (AEC, Solarbio, China), and the protein bands were analysed by AlphaView software [18 (link), 53 (link)].