Figure 1.

Construction of fbh1 mutants with substitutions in the F-box or helicase motif. (A) Schematic structure of the Fbh1 protein with the F-box and helicase domains. Sequence alignments of F-box (upper panel) and Walker A (bottom panel) motifs of S. pombe (SpFbh1), mouse (mFbh1), and human Fbh1 (hFbh1). Identical amino acids are shown in bold. Asterisks show the positions of the P15A/L26A and K301A mutations. (B) Co-immunoprecipitation of Skp1 with Fbh1. Cells expressing HA-Skp1 and GFP-Fbh1 were cultured as described in the Methods, and cellular extracts were immunoprecipitated with anti-HA antibodies. Samples were resolved by SDS-PAGE followed by western blotting with anti-GFP or anti-HA antibodies. *, non-specific bands.