Fig. 1.

Adenoviral expression of hemagglutinin (HA)-tagged Na⁺/H⁺ exchanger 3 (NHE3) in Caco-2/bbe cells. Caco-2/bbe cells were infected with purified adenovirus (Adeno)-HA-NHE3 at 0.5 × 10¹⁰, 1.0 × 10¹⁰, and 2.0 × 10¹⁰ particles/ml medium for 6–7 h (1 ml apical + 1 ml basal). Approximately 40 h after infection, cells were placed in serum-free medium for 4 h and collected for Western blot analysis, NHE3 activity assay, and immunostaining. A: Western blot analysis was performed on lysates of Caco-2/bbe cells infected with different amounts of Adeno-HA-NHE3; 40 μg of protein was used for Western blot analysis, and NHE3 expression was detected with mouse anti-HA monoclonal antibody. NHE3 expression linearly correlated with number of viral particles exposed. B: confocal microscopic x-y section (left), x-z section (top), and y-z section (right) of Caco-2/bbe monolayer infected with 2.0 × 10¹⁰ Adeno-HA-NHE3 particles/ml and immunostained with anti-HA antibody and nuclear marker (Hoechst). NHE3 expression was mostly at/near the apical surface. C: 50 μg of total cell lysate protein from Caco-2/bbe, Caco-2/bbe/Adeno-NHE3, and mouse jejunum were immunoblotted (IB) with polyclonal antibodies against NHE3 (top), monoclonal HA antibody (middle), and monoclonal β-actin (bottom). NHE3 expression was quantitated and normalized with β-actin from the same samples. Adeno-HA-NHE3 expression in Caco-2 cells (Adeno) was similar to endogenous mouse jejunum NHE3 expression, but endogenous NHE3 expression in Caco-2 cells (Endo) was negligible. AU, arbitrary units. D: cells grown on Transwell filters were infected with Adeno-HA-NHE3 and immunostained under permeabilized conditions with mouse monoclonal anti-HA antibodies and Hoechst nuclear staining. The number of NHE3-positive and -negative cells was counted with a Zeiss LSM510 confocal fluorescence microscope. The calculated rate of infection (NHE3-positive cells/total no. of cells) linearly correlated with viral particle number, with a maximum infection rate of ∼75%. Results are means ± SE from 3 experiments. E: Caco-2/bbe cells were grown on “filterslips” and infected with Adeno-HA-NHE3. Approximately 48 h later, basal NHE3 activity was measured with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)/fluorometry. Activity of NHE3 was dependent on the number of viral particles used for infection. Results are means ± SE from 3 similar experiments.