Yeast two-hybrid mating for pGBKT7-D2 and VDU1-expressing clone. (a and b) Positive control interaction for the mating: AH109 (pGBKT7-53) plus Y187 (pTD1); negative control for the mating: AH109 (pGBKT7-empty) plus Y187 (pGADT7-empty); mating: AH109 (pGBKT7-D2) plus Y187 (pGADT7-VDU1); negative control for the VDU1 mating: AH109 (pGBKT7-empty) plus Y187 (pGADT7-VDU1). (a) Mating grown on Trp^–Leu^– double-dropout (DDO) media. (b) Mating grown on Trp^–Leu^–His^–Ade^– QDO media plus X-α-Gal substrate. While all yeast strains grew on DDO media, indicating the presence of both expression plasmids, D2 did not interact with the GAL4 AD alone (data not shown), and, on QDO media (b), VDU1 did not interact with the GAL4 DBD alone, while pGBKT7-D2 interacted with the pGADT7-VDU1 clone. The top left panel indicates the growth of the positive control, while the negative control is indicated in the top right panel. The growth of PGBKT7-D2 + VDU-1 on QDO media plus X-α-Gal is shown on the lower left panel, while pGBKT7-empty + VDU-1 is shown on the lower right panel, indicating specificity of VDU-1 interaction with D2. (c) In vitro coimmunoprecipitation of GST-D2 and ³⁵S-VDU1. Crude bacterial lysates expressing GST-D2 or GST-GUS were incubated with ³⁵S-VDU1 followed by GST pulldown. Pellets were resolved by SDS-PAGE, and ³⁵S-VDU1 is indicated. Levels of GST-fusion proteins (GST-D2 and GST-GUS) were determined by Western analysis using anti-GST antibody. Approximately 1% of the input ³⁵S-VDU1 is specifically pulled down. This experiment was performed twice. (d) In vivo coimmunoprecipitation of D2 and VDU1 or VDU2. HEK-293 cells were cotransfected with FLAG-CysD2 and GFP-VDU1 or GFP-VDU2. Anti-GFP antibody was used for immunoprecipitation, and the pellets were resolved by SDS-PAGE and probed with anti-FLAG antibody by Western analysis. GFP refers to a vector containing only GFP, not fused to any other protein. This experiment was performed twice with similar results.