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.