Fig. 3.

Tul1 E3 ligase subunits form distinct subcomplexes. A, digitonin-solubilized extracts from wild-type, tul1Δ, dsc2Δ, dsc3Δ, and ubx3Δ cells were prepared, and proteins associated with Dsc2 were immunopurified by anti-Dsc2 affinity-purified antibodies. Equal amounts of input and 5× bound fractions were immunoblotted using HRP-conjugated anti-Tul1, anti-Dsc2, anti-Dsc3, and anti-Ubx3 antibodies. B, C, proteins associated with Dsc3 or Ubx3, respectively, were immunopurified as described in A using anti-Dsc3 or anti-Ubx3 affinity-purified antibodies. Equal amounts of input and 5× bound fractions were immunoblotted using HRP-conjugated anti-Tul1, anti-Dsc2, anti-Dsc3, and anti-Ubx3 antibodies. D, model illustrating the results from co-immunoprecipitation experiments in A–C. Complexes and subcomplexes observed in wild-type and mutant cells are indicated. Contacts do not denote direct protein–protein interactions.