Requirement for cytoplasmic-oriented lysine residues for AQP2 stability and trafficking.
A) Cell lines expressing a tetracycline-regulated copy of the constructs mentioned in (A) (Alexa Fluor 488; yellow) were co-stained with either αBiP (ER) or αISG75 (localises to flagellar pocket/endosome), both stained with secondary antibodies coupled to Alexa Fluor 568 (magenta). DAPI (cyan) was used to label the nucleus and the kinetoplast. Scale bars, 5 μm. B) Representative western blot (n = 3 independent replicates) of protein turnover monitored by cycloheximide (CHX) treatment followed by pulse-chase of cells expressing the constructs in (A). Cells were either untreated or treated with 25 μM MG132 for 1 h prior to harvest. Cells were harvested at 0 hours and 2 h post-CHX treatment and analysed by immunoblotting. Uninduced controls (“Un.”) were also included. C) EC50 values (average ± standard deviation; n = 3 independent replicates) of pentamidine (upper panel) and salicylhydroxamic acid (SHAM) with or without 5 mM glycerol (lower panel) following recombinant expression of either AQP2WT, AQP25K>R, or single arginine-to-lysine AQP2 mutants (AQP2R19K, AQP2R45K, and AQP2R54K). Statistical test for significance was conducted using a pairwise t test comparison with uninduced cell lines. * p<0.01, ** p<0.001, *** p<0.0001.