Fig. 3
Stability of NHE6 is diminished for ΔES mutant. a AP-1 cells were transiently transfected with NHE6v1HA WT or ΔES mutant for 24 h and then treated with 150 μg/mL cycloheximide for the indicated time points, lysed and analysed by SDS-PAGE and immunoblotting with a mouse monoclonal anti-HA (α-HAm) antibody. Equal amounts of proteins were loaded, as shown by probing the membranes with a monoclonal anti-GAPDH antibody (α-GAPDHm). b Quantitative analysis by densitometry of NHE6v1 WT and ΔES protein abundance (normalized to GAPDH levels) as a function of time in the presence of cycloheximide. Values represent the mean ± S.E.M. of three separate experiments. c-d AP-1 cells were transiently transfected with NHE6v1HA WT (c) or ΔES (d) for 24 h and then treated with 150 μg/mL cycloheximide for the indicated time points in the presence of DMSO (vehicle), the proteasomal inhibitors MG-132 (40 μM) or lactacystin (LC, 30 μM) (left panels), or the lysosomal inhibitors leupeptin/pepstatin (LeuP, 100 μg/ml) or chloroquine (CQ, 500 μM) (right panels). Cellular lysates were analysed by immunoblotting with a mouse monoclonal α-HAm antibody. Membranes were also probed with a mouse monoclonal α-GAPDHm antibody as a loading control