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Fig. 6. Isolation of HOPS binding protein by the yeast two-hybrid system. HOPS full-length protein was used as bait. (A) Growth of transformants co-expressing HOPS and eEF-1A on selective medium. HOPS, HOPS-Gal4-DBD; eEF-1A, the clone isolated from the screening of the library; Gal4DBD, VP-16-AD and lamin are negative controls. Individual Trp+ and Leu+ transformants were plated on selective medium (Trp–Leu–) with histidine and adenosine (HIS+/ADE+) or without histidine and adenosine (HIS–/ADE–). (B) ß-galactosidase assay in selected colonies of yeast expressing HOPS and eEF-1A. ß-galactosidase units are expressed in Miller units. (C) Coimmunoprecipitation of HOPS-EF-1A complex from H-35 hepatoma cells. (Top) Western blot analysis of H-35 protein extract immuno-precipitated with preimmune and anti-HOPS sera (IP). H-35, protein extract as control. Detection was performed with anti-EF-1A. (Bottom) Western blot with anti-eIF-2a as control.
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