Interaction of TsATG4B with IECs by confocal microscopy and Far-Western blot analysis. A The subcellular localization of rTsATG4B bound to IECs was observed via confocal microscopy (1000×). IECs were pre-incubated with rTsATG4B for 2 h at 37 °C, and then the same procedure used in the immunofluorescence assay was performed. Images were acquired and analysed with an Olympus FV1200 laser scanning microscope. B Analysis of rTsATG4B binding to IEC proteins by Far-Western blotting. After the IEC lysates (lanes 1–3) and C2C12 lysates (lanes 4–6) were transferred to NC membranes, the NC membranes were incubated with rTsATG4B for 2 h at 37 °C. Then, the membranes were individually probed with anti-TsATG4B serum (lanes 1, 4), infection serum (lanes 2, 5) and pre-immune serum (lanes 3, 6). Binding between rTsATG4B and IEC lysates was detected with anti-TsATG4B serum (lane 1) and infection serum (lane 2).