Fig. 5.

Effect of acid incubation on apical membrane NHE8 protein abundance and Na⁺/H⁺ exchange activity with inhibition of RNA synthesis. Apical membrane NHE8 was assayed using surface biotin labeling and streptavidin precipitation in the presence of either 5 × 10⁻⁶ M actinomycin D to inhibit mRNA synthesis or vehicle. Actinomycin D was added 1 h before incubation of cells with the different pHs for 24 h. Apical membrane NHE8 was increased in cells incubated with an acidic media in the presence of actinomycin D as shown in A. A typical immunoblot is shown below. B: effect of actinomycin D on Na⁺/H⁺ exchanger activity. Cells incubated at pH 6.6 in the presence of actinomycin D still had a faster dpH_i/dt in response to the addition of sodium after cell acidification than cells incubated at pH 7.4. A typical immunoblot is shown in C where the abundance of NHE8 is greater in cells incubated at pH 6.6 compared with 7.4.