Fig. 3.

Alpha-actinin detection on endothelial and epithelial cells. A. To determine localization of α-actinin in epithelial and endothelial cells, confluent HBMEC monolayers were fixed with paraformaldehyde and either left intact (a) or permeabilized using Triton X-100 (b). Subsequently, the monolayers were stained for α-actinin followed by FITC-conjugated secondary antibody and examined by confocal microscopy. The presence of α-actinin was only detected in permeabilized cells (b). Top views (X–Y sections) of cells are shown in a and b. X–Z section of permeabilized cells demonstrating α-actinin distributed within the cytoplasm is shown in c. An equivalent section incubated without the primary antibody is shown in d. Similar results were observed using HMEC-1, EAhy926 and Chang cell lines (not shown). B. Confluent monolayers were labelled with anti-α-actinin or anti-αvβ5 antibody and examined by flow cytometry. The α-actinin protein was not detected on the surface of any of the cell lines tested whereas the integrin was detected in all the cell lines. Secondary antibody controls (second ab only) are shown in each case. Data from one representative of two independent experiments are shown. C and D. Confluent HBMEC (C) and HMEC-1 (D) monolayers were incubated with Opc⁺ Nm of strain C751 for 3 h in the absence of normal human serum (−NHS) and in addition, either in the absence or in the presence of polyclonal anti-α-actinin antibody as shown. From viable count assays, it was apparent that in the absence of serum, Nm binding was low and not affected by the presence of the blocking antibody. In control experiments, inhibition of normal human serum-mediated bacterial adhesion (+NHS) in the absence and the presence of anti-α-actinin or anti-αvβ3 antibody was also performed. The anti-integrin antibody inhibited surface attachment significantly even at 10 μg ml⁻¹ but anti-α-actinin antibody had no effect. Means and standard errors of two experiments performed in triplicates are shown. *P-value ≤ 0.05% as determined using Student's t-test.