Anti rabbit igg

The lung tissue lysate was precleaned by adding 10 μL of Protein A/G Plus Agarose (Sigma, Merck KGaA, Darmstadt, Germany) and incubated at 4 °C for 1 h. Rabbit anti-ACE2 monoclonal antibody (Proteintech, Chicago, USA), rabbit antiblood group A antibody (ARP), or antirabbit IgG (Genetex, CA, USA) was mixed with Protein A/G Plus Agarose and incubated at 4 °C for 4 h. The antibody–agarose mixtures were individually mixed with precleaned tissue lysate and incubated at 4 °C overnight. After incubation, agaroses were washed 5 times with lysis buffer and the bound proteins were eluted with SDS-PAGE sample buffer.

The GM1 enzyme-linked immunosorbent assay was used to determine the CT production in culture supernatant (Lee et al., 2012 (link)). Purified whole CT (Cayman Chemical, Ann Arbor, MI, United States) was used to provide a standard curve. Rabbit polyclonal anti-CTB (GeneTex, Irvine, CA, United States) and anti-rabbit IgG (GeneTex) were used for detection. The optimal laboratory conditions for CT production from classical biotype strains were as follows: culture at 30°C, LB pH 6.5 buffered with 50 mM Tris–which has been described as “agglutinating condition” (Waldor and Mekalanos, 1996 (link)). CT production (amount of CT/1 OD₆₀₀) in the classical biotype strain O395 under such conditions was considered the reference CT production value. CT production from El Tor strains was measured in culture media after 16 h of single-phase shake culture at 30 and 37°C in LB, PBS-buffered LB, AKI media (0.5% NaCl, 0.3% NaHCO₃, 0.4% yeast extract, and 1.5% Bacto-Peptone), and LB buffered with 50 mM Tris pH 6.5. CT production (amount of CT/1 OD₆₀₀) values of mean ± standard deviation in El Tor strains were obtained from three independent experiments and were compared with the reference value. Expression of CT in V. cholerae strains was additionally confirmed by immunoblot analysis using anti-CT (Supplementary Figure S1).