Fig. 4. Functional Sly1 protein promotes or stabilizes the Bet1-Sed5 complex in vitro. The sly1^ts strain was transformed with a recombinant 6myc-SED5 gene under the SED5 promoter on a CEN plasmid (I) or with a 3HA-BET1 gene under the GAL1 promoter on a 2µ plasmid (II). Preparation of the yeast lysates and recombinant proteins are described in Materials and Methods. The reaction time was fixed at 20 minutes, but the reaction temperature was 0°C (lanes 1 and 2), 15°C (lanes 3 and 4), 25°C (lanes 5 and 6) or 35°C (lanes 7 and 8). At the end of the reaction, the mixture was diluted and solubilized with Triton X-100 and subjected to immunoprecipitation using an anti-myc antibody. Western blotting was done to detect 6myc-Sed5, 3HA-Bet1 and Sly1/Sly1^ts-Strep and endogenous Sly1^ts protein in lysate I and II. The bands of Sly1/Sly1^ts-Strep, 6myc-Sed5 and 3HA-Bet1 are indicated by arrowheads. ^*indicates the endogenous Sly1^ts protein in lysate I and II.