Fig. 5. Mob2p interacts with Orb6p directly. (A) ß-galactosidase assays to test for interaction between Mob2p and Orb6p. Strains carrying plasmids expressing Orb6p, Mob2p, Snf1p or Snf4p fused to the Gal4p DNA-binding domain (GBD) or activation domain (GAD) were tested as indicated (see Materials and Methods). Assays are shown in duplicate. (B) Co-immunoprecipitation of Mob2p-13Myc and Orb6p-3HA. orb6-3HA cells (YDM1077), mob2-13myc cells (YDM975) and cells containing both tagged genes (YDM1151) were grown to exponential phase. The same concentration of whole cell lysates was used. Anti-HA immunoprecipitates were immunoblotted with anti-Myc (top panel) and anti-HA (bottom panel). (C) In vitro binding assay for Mob2p and Orb6p. In vitro synthesized [³⁵S] methionine-labeled 2Myc-6His-tagged Mob2p (MH-Mob2p) and 3HA-tagged Orb6p (HA-Orb6p) either alone or mixed together were incubated for 30 minutes at 30°C, then subjected to immunoprecipitation with anti-Myc antibodies and resolved by SDS-PAGE (top panel). The HA-Orb6p level in each sample was detected by anti-HA immunoprecipitation (bottom panel). The images were obtained by fluorography. Note that both HA-Orb6p and MH-Mob2p run as 2 and 3 bands, respectively, which most probably result from protein degradation or premature termination of transcription or translation.