FIGURE 2.

Elevated Ca²⁺ (ionophore 4-Br-A23187) caused a rapid and prolonged decrease in NHE3 surface expression. A, OK/NHERF2/HA-NHE3 cells were treated with 4-Br-A23187 (0.5 μm) for 0, 2, 10, and 30 min at 37 °C in a 5% CO₂, 95% air atmosphere, and the cells were then quickly washed with cold PBS three times (4 °C) and kept at 4 °C for 30 min. Cells were then biotinylated, and the total lysate was collected in 1 ml. The surface-biotinylated proteins were collected by avidin precipitation (surface) and retrieved in 0.1 ml of sample buffer. The NHE3 protein was loaded into each lane with the same volume in a series of dilutions (10, 20, and 30 μl), which were detected by monoclonal anti-HA antibody. Results from a single experiment are shown. B, results from three experiments as in A are shown as mean ± S.E. In each experiment, results were normalized with time 0 set as 100%. p values are compared with untreated control (paired t test). NS, not significant.