FIGURE 4.

AUP1 is both ubiquitylated and binds ubiquitin-modified proteins, and anti-AUP1 immunoprecipitates are able to perform ubiquitin transfer in vitro. A, HeLa cells were transfected with HA-ubiquitin and empty vector or one of the GFP-AUP1 constructs (WT or mutants, as indicated). HA-Ub was recovered with 3F10 (HA-specific) antibody from digitonin lysates supplemented with 2.5 mm N-ethylmaleimide. GFP-AUP1 content in total cell lysates and immunoprecipitates (IP) was determined by immunoblotting (IB) with an anti-AUP1 antibody. B, GFP-AUP1 was recovered with anti-GFP antibody from lysates described in A. HA-Ub content in total cell lysates and immunoprecipitates was determined by immunoblotting with an anti-HA antibody. C, HeLa cells were transfected with empty vector or one of the HA-AUP1 constructs (WT or mutant, as indicated). HA-AUP1 was immunoprecipitated from digitonin lysates. E1 (100 nm), FLAG-ubiquitin (60 μm), and an ATP-regenerating buffer was added to the immunoprecipitates and kept at 37 °C for 60 min. Separate samples containing only FLAG-ubiquitin and buffer or E1, FLAG-ubiquitin, and buffer served as controls. Samples were run on an 8% Tris-Tricine SDS-polyacrylamide gel, and were immunoblotted with a FLAG-specific antibody.