TIR1 interacts with the Skp1-like proteins ASK1 and ASK2. (A) Sequence alignment of the A. thaliana ASK1 and ASK2 (GenBank accession no. AF059295) proteins as well as human and S. cerevisiae Skp1p with PileUp (Genetics Computer Group, Inc., Madison, WI). Identical amino acids are boxed in black. Similar amino acids are boxed in gray. (B) Serial dilutions of yeast strain YPB2 carrying the indicated two-hybrid plasmids. Cells were plated onto 3-AT selection medium (left) or control (right) medium and incubated at room temperature. The 3-AT plate was photographed after 10 days and the control plate after 3 days. (C) Western blot analysis of seedling extracts with polyclonal antisera raised against ASK1 (lanes 2–4) or ASK2 (lanes 6–8). (D) Western blot analysis of crude seedling extracts (lanes 1,2,5,6) or α-myc immunoprecipitates (lanes 3,4,7,8). (Bottom lanes 1–4) was probed with α-ASK1 antisera. (Bottom, lanes 5–8) Probed with α-ASK2 antisera.