Fig. 4.

Total and surface NHE3 levels were increased under basal conditions in myosin VI KD Caco-2/Bbe cells. (A,B) Western blot detection and quantification of total NHE3 in myosin VI KD cells. Cells were grown in six-well plates and infected with Ad-HA-NHE3 at day 12. At 48 h after infection, cells were processed for western blotting. Total NHE3 amount was normalized with β-actin. Results are mean±s.e.m. of three independent experiments. In myosin VI KD cells, the total NHE3 amount was significantly increased (P<0.05). (C) NHE3 expression in myosin VI KD cells as detected by immunofluorescence (confocal microscopical) analysis. Cells were filter grown for 12 days, infected with Ad-HA-NHE3 and 2 days later processed for antibody staining. Cells were not initially permeabilized and were surface labeled with Alexa-Fluro-568-conjugated WGA at 4°C; then cells were permeabilized and labeled with anti-HA antibody. (C) Compared to control cells (a1, a2, a3), NHE3 expression was dramatically increased in myosin VI KD cells (b1, b2, b3). Scale bar: 20 µm. (D) Statistical analysis of total NHE3 intensity by Volocity software. The total NHE3 amount was increased (P<0.05) in myosin VI KD cells. Results are mean±s.e.m. of ten images from three independent experiments. (E) Quantification of the percentage of NHE3 on the surface by Volocity software. The percentage of surface NHE3 was increased (P<0.05) in myosin VI KD cells. Results are mean±s.e.m. of 15 images from three independent experiments.