TbAQP2 transits through the endosomal compartment and is efficiently delivered and degraded in the lysosome.
A) Cell lines expressing a tetracycline-regulated copy of 3xHAAQP2 (Alexa Fluor 488; yellow) were co-stained with anti-TbRab5a and anti-TbRab5b (early endosomes), anti-TbRab11 (recycling endosomes), and anti-p67 (lysosome). All endosomal and lysosomal markers were labelled with secondary antibodies coupled to Alexa Fluor 568 (magenta). DAPI (cyan) was used to label the nucleus and kinetoplast. Scale bars 5 μm. A schematic depiction of the results from confocal microscopy is included in the right panel, generated with BioRender. B) Left panel; Protein turnover was monitored by cycloheximide (CHX) treatment. Cells were harvested at various times and the protein level monitored by immunoblotting. ISG75 was included as a control. Right panel; Quantification for ISG75 and 3xHAAQP2. Results are the mean ± standard deviation of three independent experiments. C) Upper panel; As in (B), but cells were untreated or exposed to 100 nM of bafilomycin A1 (BafA1), or to 25 μM of MG132 for 1 h prior to harvesting. Cell lysates were resolved by SDS-PAGE followed by western immunoblotting using anti-HA antibody. Lower panel; Quantification from three independent experiments—dotted line represents 100% (signal at 0h). Data presented as mean ± standard deviation (n = 3 independent replicates). Statistical analysis was conducted using t test; * p<0.01 and the signal from untreated cells at 1 h as reference.