FIGURE 9.

POSH stimulates ubiquitination of ROMK. A, in vitro ubiquitination assay shows the effect of POSH on ROMK1 ubiquitination. ROMK proteins (generated with TnT quick coupled transcription/translation system) were incubated at 37 °C for 30 min with exogenous ubiquitin, ATP, E1 and E2 ubiquitin ligase (UbcH5a) in the presence or absence of His-tagged POSH. After the reaction ROMK proteins were resolved by electrophoresis and Western blotting with either ROMK or ubiquitin antibodies. B, a Western blot shows the effect of POSH or POSHΔRING on ROMK1 ubiquitination. 293T cells were transfected with ROMK1 (R1), R1+POSH, or R1+ POSHΔRING. MG132, an inhibitor of proteosome was added during experiments. IP, immunoprecipitation; IB, immunoblot.