Figure 4.

Constitutive expression of EspF and Map depletes tight junction proteins. (A) Cell lysates derived from untransfected, EGFP, EGFP-Tir, EGFP-EspF and EGFP-Map cell lines were separated by electrophoresis on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. Equal amounts of cell lysates were loaded after normalizing with GAPDH. Blots were probed with the indicated primary antibodies and HRP-conjugated secondary antibodies. Full-length blots are presented in Supplementary Figure S3. (B,C) The x-ray films were scanned and quantitative analysis was performed by measuring band intensities using ImageJ software. The expression of different tight junction proteins in these cell lines was normalized with respect to untransfected cells and fold changes were calculated. Data are represented as means ± s.e.m. from six independent experiments using two biological replicates for all cell lines; **represents p-values < 0.001. (D) The cell surface proteins were labeled with membrane impermeable Sulfo-NHS-Biotin and immobilized on streptavidin-agarose beads. The beads were washed and subjected to SDS-PAGE. E-cadherin was used as a loading control for cell surface proteins. Full-length blots are presented in Supplementary Figure S4. (E) The levels of cell surface proteins were estimated by densitometric scanning of x-ray films using ImageJ software. Shown are fold changes with respect to untransfected cells. Error bars represent means ± s.e.m. from three independent experiments; **represents p-values < 0.001; UT: Untransfected.