Fig. 5.

The surface NHE3 amount was not changed by FSK and CCH in myosin VI KD Caco-2/Bbe cells but was decreased in control cells. Cells were grown in six-well plates and infected with Ad-HA-NHE3 at day 12. After 48 h, cells were serum-starved for 4–5 h then treated with 25 µM CCH for 5 min or 10 µM FSK for 15 min at 37°C before assessing the amount NHE3 by a cell surface biotinylation assay. Detection and quantification of total and surface amounts of NHE3 are shown in A and B (single experiments), and C and D (mean±s.e.m. of three independent experiments). Two dilutions of total, surface and cytosol fractions were loaded and the surface expression of NHE3 was determined as a percentage of total NHE3 and normalized to β-actin; the values for the controls were set to 100 in each experiment. As shown in A and C, CCH- and FSK-treated control cells expressed ∼40% less surface NHE3 than non-treated cells (P<0.05). In myosin VI KD cells, there was no significant (NS) change of surface NHE3 expression after treatment with CCH or FSK compared to non-treated cells (B,D).