TbAQP2 is ubiquitylated in T. brucei.
A) Cells expressing 3xHAAQP2 were treated with either NH4+Cl (10 mM) or MG132 (25 μM) for 1h prior to harvesting. Cell lysates were resolved in a 4–12% acrylamide gel and detected with anti-HA antibody by western blotting. The intensity of anti-β tubulin was used as loading control. B) Immunoprecipitation of Δaqp or 3xHAAQP2 cell lysates with anti-HA beads followed by anti-ubiquitin detection by western blotting. An anti-HA blot was also included to confirm protein expression upon induction with tetracycline. Anti-β tubulin was used as loading control. ‘*’ indicates the predicted migration poition of a monoubiquitylated 3xHAAQP2. C) As in (B), but immunoprecipitation conducted using ubiquitin capture matrix and analysed by western blotting (left panel). The total (“T”), unbound (“Unb.”), wash (“W”), and elution (“E”) fractions were resolved by SDS-PAGE electrophoresis and analysed with anti-HA immunoblotting (right panel). ‘*’ is as in panel B.