Rabbit anti laminin

Immunofluorescent staining was conducted to evaluate
the component of MEF-ECM. The MEFs and MEF-ECM
were firmly fixed with a 4% paraformaldehyde solution for
15 minutes at room temperature. To facilitate permeability,
the specimens were treated with a 0.1% Triton X-100
solution for 10 minutes. Subsequently, the specimens
were blocked using a 1% bovine serum albumin (BSA,
Sigma, USA) solution for the next 30 minutes. Next,
primary antibodies including rabbit anti-laminin (1:100,
Invitrogen, USA), collagen I (1:300, BD Biosciences,
USA) and fibronectin (1:200, BD Biosciences, USA)
were utilized to incubate MEF-ECM samples overnight
at 4˚C. The MEF-ECM specimens underwent incubation
with Alexa Fluor-488 conjugated goat anti-rabbit (1:300,
Invitrogen, USA) in a dimly lit environment for an hour at
ambient temperature. The MEF samples were incubated
with Alexa Fluor-568 conjugated phalloidin (1:1000,
Cell Signaling Technology, USA) for 30 min at dark.
Subsequent to the completion of the staining process,
4,6-diamidino-2-phenylindole (DAPI, Invitrogen, USA)
was utilized to counterstain all samples within 10 minutes,
after which images were obtained under an inverted
fluorescence microscope (Leica Microsystems, Wetzlar,
Germany).

Protein expression profiling was carried out using extraction of Non-Infected cells (NI) and cells infected by STM-ZT, STM-Z, STM-T or STM-3d strains. Bacteria were deposited on IEC-6 with a multiplicity of infection of 10, for 1.5 h, followed by gentamicin (100 µg/ml) for 1.5 h. Cells were then resuspended in 100 µL of Laemmli buffer and denaturated 10 min at 100°C. Whole-cell protein samples 25 µL were run on SDS- PAGE (100 V) in a 4–15% Miniprotean TGX Precast Protein gels (Bio-Rad) in a Tris-glycine running buffer (25 mM Tris base, 192 mM glycine, 0.1% [wt/vol] SDS [pH 8.31]) and transferred onto a nitrocellulose membrane with Trans-blot Turbo transfer System (Bio-Rad) in Tris-glycine buffer system 15 min at 25 V and 2.5 mA. The blots were probed with the first antibody (1:1000), rabbit anti-laminin (Invitrogen), rabbit anti-fibronectin (Invitrogen) or mouse anti-tubulin overnight at 4°C and detected by chemiluminescence using goat anti-rabbit secondary antibody (1:25000) (Pierce) or goat anti-mouse secondary antibody (1:5000) (Dako) conjugated to HRP for 1 h. Proteins were revealed using the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific).