FIG. 3.

Mapping of interacting domains of pp28 for the self-interaction by pulldown assays. (A) Schematic diagram of the GST-pp28 fusion proteins depicting the primary structure of the deletion mutants. Shaded horizontal bars indicate the residues expressed by the mutants. The pp28 binding activity of each mutant is presented at the right of the figure: +++, activity 100% of the wild type; ++, activity 70 to 90% of the wild type; +, activity <10% of the wild type; and −, activity 0% of the wild type. (B) SDS-PAGE analysis (top) showing the amounts of GST-pp28 fusion proteins on beads prior to pulldowns, and immunoblots (middle and bottom) showing antibody specificity of GST-pp28 fusion proteins on beads. Glutathione-conjugated beads with associated proteins were subjected to SDS-PAGE and visualized by Western blotting with anti-pp28 MAb and anti-BB tag MAb as described in Materials and Methods. Note that GST-pp28Δ26-33 and GST-pp28Δ26-43 were not detected by anti-pp28 MAb, indicating that the epitope recognized by this MAb included aa 26 to 33 of pp28. (C) Immunoblots showing the pp28 binding activity of deletion mutants. Equal amounts of the purified GST alone or GST-pp28 fusion proteins on beads in panel B were incubated with lysate from bacteria expressing BB tag-pp28. After extensive washing, bound proteins were subjected to SDS-PAGE and visualized by Western blotting with anti-pp28 MAb 41-18 and anti-BB tag MAb 28-19 as described above. The input lane shows 10% of the amount of the BB-pp28 fusion protein lysate that was added to the binding reaction.