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Figure S1. Clp1 and Mid1 interact in vivo. (A) nda3-KM311, nda3-KM311 clp1-GFP, nda3-KM311 mid1-myc, and nda3-KM311 clp1-GFP mid1-myc strains were arrested by incubation at 18°C for 7 h, samples were collected, and native extracts were prepared. After immunoprecipitation with 3E6 (GFP) and MYC antibodies, samples were separated by SDS-PAGE and detected by Western blot analysis with MYC and GFP (Roche) antibodies. (B, top) Schematic representation of Mid1 depicting the following protein domains: NES (nuclear export signal); PEST, proline (P), glutamate (E), serine (S), and threonine (T)–rich domain; HLX (amphipathic helix motif); NLS; and PH (pleckstrin homology domain). (B, bottom) Yeast two-hybrid analysis of Mid1 fragments against Clp1 and Clp1 phosphatase-dead (C286S) protein. Cells cotransformed with either bait plasmid pGBT9:Clp1 or pGBT9:C286S and the depicted Mid1 fragments in prey plasmid pGAD424 were analyzed for growth (+ or −) on -HIS -ADE plates. (C) Recombinant MBP and MBP-Clp1 bound to amylose beads were incubated with GST-Mid1(331–534) fusion protein, washed extensively, and then separated by SDS-PAGE followed by Coomassie blue staining (top) or Western blot analysis (bottom) with GST antibody. Input contains 10% of the GST-Mid1(331–534) included in reactions. (D, top) Schematic representation of Clp1 protein domains. PTP, protein tyrosine phosphatase domain. A and B domains are based on amino acid sequence homology to human Cdc14B and Saccharomyces cerevisiae Cdc14. (D, bottom) Yeast two-hybrid analysis of Clp1 fragments against Mid1(331–534) as described in Fig. 1 B. (E) GST-Mid1(331–534) fusion protein bound to glutathione beads were incubated with the indicated MBP fusion proteins, washed extensively, and then separated by SDS-PAGE followed by Western blot analysis (top )with anti-MBP antibody or Coomassie blue staining (bottom). (F) Native extracts were prepared from nda3-KM311 clp1-MYC and nda3-KM311 clp1-MYC mid1Δ cells after arrest by incubation at 18°C for 7 h and then incubated with MYC antibodies to immunoprecipitate Clp1-MYC. Immunoprecipitates were treated with λ phosphatase, separated by SDS-PAGE, and detected by Western blot analysis with MYC antibodies.