Figure 3.
AIP4 polyubiquitinates itself, as well as Deltex, by K29-linked chains. (A) Ubiquitination of Deltex (DTX). HEK293T cells were transfected with Myc-tagged DTX, AIP4 and vesicular stomatitis virus (VSV)-tagged ubiquitin (Ub) expression vectors. The number indicates the only lysine residue remaining in the ubiquitin molecule. DTX immunoprecipitates (IP Myc, lanes 1–9) or ubiquitin immunoprecipitates (IP VSV, lanes 10–13) and the corresponding extracts were analysed by successive immunoblotting with antibodies against VSV, Myc and pan-cadherin. (B) Stabilization of ubiquitinated DTX by lysosomal inhibitors. 293T cells were treated with MG132 or leupeptin and NH4Cl (Leu+NH4Cl) for 2 and 48 h after transfection. Extracts were analysed as described above. WB, western blot.