For the detection of SggEsxA, TX20005 and TX20005Δesx were grown in BHI at 37°C with shaking for ~18 hours. Cultures were centrifuged, supernatants collected, filtered through a 0.2 μm filter, and concentrated using the Pierce™ Protein Concentrator PES, 3K Molecular Weight Cutoff. Bacterial pellets were washed and resuspended in PBS. Supernatants and bacterial suspensions were boiled in SDS loading dye and subjected to SDS-PAGE. Membranes were probed with anti-EsxA rabbit serum (1:100) (Pacific Immunological) and anti-FtsZ (1:250) antibodies (Abbexa, cat no. abx319936), followed by incubation with secondary antibodies conjugated to horse radish peroxidase (HRP) (1:3000). Blots were washed and immersed in Clarity Max ECL (Bio-rad). Images were acquired using a Fluorchem M chemiluminescent imager (Protein Simple).
Detection of β-catenin and PCNA was carried out as described previously [12 (link)]. Briefly, total cell lysates from HT29 cells cultured in the presence or absence of bacteria or CS were subjected to SDS-PAGE, transferred, and probed with antibodies against β-catenin (1:1000), PCNA (1:1000), and β-actin (1:1000) (Cell Signaling Technology), followed by incubation with HRP-conjugated secondary antibodies (1:3000). Band intensities were measured using ImageJ and normalized to that of β-actin.
Detection of β-catenin and PCNA was carried out as described previously [12 (link)]. Briefly, total cell lysates from HT29 cells cultured in the presence or absence of bacteria or CS were subjected to SDS-PAGE, transferred, and probed with antibodies against β-catenin (1:1000), PCNA (1:1000), and β-actin (1:1000) (Cell Signaling Technology), followed by incubation with HRP-conjugated secondary antibodies (1:3000). Band intensities were measured using ImageJ and normalized to that of β-actin.
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