α zo 1

The CamAlstR-C was fused with SYFP on its C terminus and expressed in Huh7 cells. The cells were fixed in 4% paraformaldehyde (PFA) on ice for 15 min. Blocking was performed in 2% bovine serum albumin (BSA) for 1 hr at room temperature (RT). In order to mark the cell membrane α-ZO1 (Invitrogen, catalog number: 40–2200) was used in a dilution of 1:200 in 2% BSA. The secondary antibody was Alexa 555-conjugated α-rabbit IgG and used in 1:200 dilution in 2% BSA. Nuclei were stained with TO-PRO^®-3 (Thermo Scientific) according to the manufacturer’s instructions. The samples were imaged and analyzed on a LEICA-SP5 confocal microscope (TCS, Leica, Germany).

Cells were grown on Nunc Thermanox coverslips (Thermo Fisher Scientific), inoculated with STm (MOI 10) for 1 h and fixed with 4% paraformaldehyde. Tight junction proteins were visualized by staining with a primary mouse α-occludin or rabbit α-ZO-1 (2 μg/mL; Invitrogen) followed by secondary goat α-mouse Alexa Fluor 488 or goat α-rabbit Alexa Fluor 594 (Invitrogen). Nuclei were counterstained with DAPI and cells imaged using a Zeiss 510 Meta confocal microscope.

For EhADH immunodetection, we obtained rabbit polyclonal antibodies (α-EhADH) against a specific EhADH peptide (N-566 QCVINLLKEFDNTKNI 582-C) localized within the adherence domain. New Zealand male rabbits were immunized three times (each 2 weeks) with 300 μg of this peptide diluted in TiterMax® Gold Adjuvant liquid (Sigma). Other primary antibodies used were: mouse against EhADH (mAbAdh) (Arroyo and Orozco, 1987 (link)), α-actin (kindly donated by Dr. José Manuel Hernández from Department of Cellular Biology, CINVESTAV, Mexico), α-claudin-1 (Invitrogen), α-occludin (Invitrogen) and α-caveolin-1 (Santa Cruz Biotechnology); rabbit α-ZO-2 (Invitrogen), α-ZO-1 (Invitrogen), α-occludin (Invitrogen), α-α/β tubulin (Cell Signaling), and α-PCNA (Azuara-Liceaga et al., 2018 (link)); and goat α-clathrin (Santa Cruz Biotechnology) and α-GAPDH (Santa Cruz Biotechnology). For some experiments, mouse IgM isotype control (Thermo Fisher) was used. Secondary antibodies included: α-rabbit, α-mouse and α-goat HRP-labeled IgG (1:10,000) (Life technologies); and α-rabbit, α-mouse and α-goat FITC-, TRITC- and Cy5-labeled IgM, and IgG (1:100) (Zymed) antibodies.