Western blot was performed as previously described.24 Chlamydial proteins were co-expressed with the c-Myc tag under the same inducible promoter. Thus, the detection of c-Myc was inferred as foreign chlamydial protein expression. The membrane was probed with 1:500 anti-c-Myc antibody (BioLegend, San Diego, CA, USA) and 1:1,000 anti-mouse IgG-AP-linked antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) as a secondary antibody. The intensity of the specific bands was visualized with 1-step NBT/BCIP (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) substrate solution (Thermo Fisher Scientific).
Western blot was also performed to detect the presence of flagellar proteins in the bacterial samples. The membrane was probed with 1:1,000 anti-E. coli Flagella H Pool A serum (Statens Serum Institut, Copenhagen, Denmark) as the primary antibody and 1:1,000 anti-rabbit IgG-AP antibody in TBST (Sigma-Aldrich, St Louis, MO, USA) as the secondary antibody. The intensity of the specific bands was visualized with a 1-step NBT/BCIP substrate solution (Thermo Fisher Scientific).
Western blot was also performed to detect the presence of flagellar proteins in the bacterial samples. The membrane was probed with 1:1,000 anti-E. coli Flagella H Pool A serum (Statens Serum Institut, Copenhagen, Denmark) as the primary antibody and 1:1,000 anti-rabbit IgG-AP antibody in TBST (Sigma-Aldrich, St Louis, MO, USA) as the secondary antibody. The intensity of the specific bands was visualized with a 1-step NBT/BCIP substrate solution (Thermo Fisher Scientific).