Figure 2
Developing an immunoglobulin A (IgA) transcytosis assay. (a) Wild-type (WT) and pIgR−/− cells were seeded in Transwells and treated with ±10 μM DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester)±1 μg ml−1 lipopolysaccharide (LPS) as indicated. On day 3 after seeding, 40 μl of normal mouse IgA (Santa Cruz Biotechnology) was added to the lower compartment of the Transwells, and supernatants from the upper compartment were taken at 6 h for the detection of IgA by enzyme-linked immunosorbent assay (ELISA). (b) Time-course and (c) IgA dose–response curve experiments were performed on WT and pIgR−/− cells treated with 10 μM DAPT and 1 μg ml−1 LPS to determine the optimal conditions for future experiments. An LPS dose–response curve was performed to determine whether (d) IgA transcytosis and (e) pIgR expression were dose-dependent. All LPS-treated cells were also treated with 10 μM DAPT. For IgA transcytosis, results from the ELISA were normalized to the 1 μg ml−1 LPS treatment group (=100%). Gene expression analysis by quantitative real-time polymerase chain reaction for pIgR was performed by normalizing to glyceraldehyde 3-phosphate dehydrogenase, and data are presented as fold change relative to untreated cells. The dotted lines represent the limit of detection by the ELISA. All values are indicated as mean±s.e.m. One-way analysis of variance: (a) F=573.3, P<0.0001, n≥3 per group; (b) F=539.2, P<0.0001, n≥12 per group; (d) F=675.7, P<0.0001, n≥3 per group; and (e) F=46.22, P<0.0001, n≥6 per group. Means with different letters are significantly different by Bonferroni’s multiple comparison test. ND, not detected; pIgR, polymeric Ig receptor.
