FIGURE 3.

Arsenic targets CFTR for lysosomal degradation. A, to determine whether arsenic promoted the lysosomal degradation of CFTR, CFBE cells were treated with vehicle (water) or arsenic (10 ppb) for 2 h in the presence of lysosomal or proteasomal inhibitors, and CFTR in cell lysates was detected by Western blot analysis. Arsenic reduced the amount of CFTR, confirming studies reported in Fig. 2A. Chloroquine (CHQ; 200 μm) and ammonium chloride (NH₄Cl; 50 mm), inhibitors of the lysosomal degradation of proteins, blocked the arsenic-induced decrease in CFTR abundance. Lactacystin (Lacta; 50 μm), an inhibitor of the proteasomal degradation of proteins, had no effect on the arsenic-induced decrease in CFTR abundance. n = 4/group.*, p < 0.05 versus control. B, to determine whether arsenic redirects CFTR from the recycling pathway to the lysosomal pathway, CFBE cells were treated with 10 ppb of arsenic for 4 h, and intracellular vesicle isolation and co-immunoprecipitation studies were conducted to determine the subcellular location of CFTR in cells treated with or without arsenic. Intracellular vesicles were purified with density gradient centrifugation, CFTR-containing vesicles were immunoprecipitated using a monoclonal CFTR antibody (clone M3A7, Upstate Biotech Millipore), and endosomes were identified by SDS-PAGE and Western blot analysis. Early endosomes were identified with Rab5a, recycling endosomes were identified with Rab11a, and late endosomes were identified with Rab7a. Data are presented as the amount of CFTR in each endosomal compartment normalized for the total amount of CFTR immunoprecipitated. n = 3/group. *, p < 0.05.