Effects of blocking the endosomal-lysosomal degradation pathway.

(A) Immunoblotting analyses of cell lysates from CLC-1-expressing HEK293T cells subject to treatment with increasing concentrations of NH4Cl for 24 hrs. (B) Quantification of CLC-1 protein expression levels in response to 24-hr treatment with different NH4Cl concentrations. Protein densities were normalized with respect to those for WT with no drug treatment by following the same procedure as described in Figure 4B. (C) Immunofluorescence images of HEK293T cells expressing myc-tagged A531V channels in the presence of 50 mM NH4Cl for 24 hrs. Cells were fixed under the permeabilized configuration. Scale bar = 10 µm. (D) Averaged instantaneous I-V curves of WT CLC-1 recorded under the cell-attached configuration. HEK293T cells were incubated in the absence (black circles; n = 15) or presence (red squares; n = 17) of 50 mM NH4Cl for 24 hrs. (E) Whole-cell current density (at −140 mV) of the A531V mutant recorded from HEK293T cells with or without the NH4Cl treatment.