Anti his monoclonal antibody

Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) antibody and anti-His monoclonal antibody were purchased from Abcam (Cambridge, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) was purchased from Promega Corporation (Madison, WI, USA). Anti-glutathione S-transferase (GST) monoclonal antibodies were purchased from EARTHOX Life Sciences (Millbrae, CA, USA). Annexin V, annexin-V-binding buffer, and PE anti-human CD235a (glycophorin A) antibodies were purchased from BioLegend (San Diego, CA, USA). DAPI (4′,6-diamidino-2-phenylindole), fluo-4, and propidium iodide were purchased from Sigma (St. Louis, MO, USA). Zaragozic acid A trisodium salt (ZA) was purchased from ChemCruz (Huissen, The Netherlands).
Recombinant toxins, 6×His-tagged ETX, GST-tagged ETX, mScar-ETX (mScarlet fluorescent protein fusion protein), CPB, Hla, and Net B proteins, were expressed and purified as previously described [32 (link),54 (link)].

To verify the ICs, the protein extract was analyzed using 10% SDS-PAGE, which was electrotransferred to a PVDF membrane. After blocking overnight with PBST containing 4% skim milk, the membranes were incubated using anti-F5 mouse serum (1:150) and anti-his monoclonal antibody (1:1000) (Abcam, Cambridge, UK) as a primary antibody. Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (1:10,000, Immunoway, San Diego, CA, USA) was used as the secondary antibody. After washing with PBST, the membrane was treated with a hypersensitive luminescent solution for observation. For the SIgA–ETEC F5 analysis, a mixture containing 80 μL of the elution products and a 20 μL 5 × loading buffer (containing 10% SDS and 5% β-mercaptoethanol) was placed in a boiling water bath for 10 min to ensure complete dissociation of the complex. The SDS-PAGE was performed and the proteins in gels were transferred to the PVDF membrane which was incubated using both antibodies of anti-F5 mouse serum (1:150) and goat anti-mouse IgA alpha chain (1:1000, ab97231, Abcam, Cambridge, UK) for 2 h at 37 °C. The membrane was washed with PBS three times and then incubated with goat anti-mouse IgG antibodies labelled with HRP and rabbit anti-goat-HRP at 37 °C for 1 h.

Anti‐MAL polyclonal antibody (reactivity: mouse, rat, dog, human, frog), anti‐ceramide polyclonal antibody, horseradish peroxidase (HRP)‐coupled goat antimouse IgG (H + L) antibody, anti‐His monoclonal antibody and fluorescein isothiocyanate (FITC)‐conjugated goat anti‐rabbit IgG (H + L) were purchased from Abcam (Cambridge, MA, USA). 3‐(4, 5‐dimethylthiazol‐2‐yl)‐5(3‐carboxymethoxyphenyl)‐2‐(4‐sulfopheny)‐2H‐tetrazolium inner salt (MTS) was purchased from Promega Corporation (Madison, WI, USA). Anti‐glutathione S‐transferase (GST) monoclonal antibody was purchased from EARTHOX Life Sciences (Millbrae, CA, USA). Annexin V, Annexin V binding buffer and PE anti‐human CD235a (Glycophorin A) antibody were purchased from BioLegend (San Diego, CA, USA). Fluo‐4 and PKH26 Red Fluorescent Cell Linker Kit were purchased from Sigma (St. Louis, MO, USA). BAPTA‐AM, Protease inhibitor and 2ʹ,7ʹ‐Dichlorofluorescin Diacetate were purchased from Sigma (St. Louis, MO, USA).