Figure 1

Atrophin-1-interacting protein 4 (AIP4) does not ubiquitinate spartin. (a) HeLa cells were transfected with Myc-wild-type spartin together with an empty vector (lane 1), hemagglutinin (HA)-ubiquitin (lane 2), HA-ubiquitin and Flag-AIP4 (lane 3), or HA-ubiquitin and Flag-AIP4C830A (lane 4). TCL = total cell lysates. WB = western blot. An asterisk (*) indicates crossreactive proteins. Sizes of protein standards are indicated to the left in kDa. (b) Immunoprecipitates of HA-wild-type, mutant PY/AA, or 211-666 fragments of spartin and Flag-ubiquitin analyzed by immunoblotting with anti-Flag (upper panel) or anti-HA (lower panel) antibodies. (c) HeLa cells were transfected with Flag-AIP4 alone or together with HA-wild-type spartin, HA-spartin PY/AA, or HA-spartin 211-666 fragment. Cell lysates were immunoprecipitated (IP) with anti-HA antibodies and immunoblotted with anti-Flag (upper panel) or anti-HA (lower panel) antibodies. An asterisk (*) and kDa are indicated as in A. (d) Schematic diagram of spartin constructs and their status of ubiquitination and binding to AIP4.