Fig. 5. Time course of Bet1-Sed5 complex formation with Sly1 or Sly1^ts. (A) The cell-free assay system was carried out at the various reaction times indicated. Yeast lysate and a recombinant protein were mixed as described in Fig. 4. The reactions were started by shifting the temperature to 35°C and were continued for up to 20 minutes. The bands of Sly1/Sly1^ts-Strep, 6myc-Sed5 and 3HA-Bet1 are indicated by arrowheads at the right. ^*indicates the endogenous Sly1^ts protein in lysate I and II. (B) Reactions were incubated for 20 minute at 0°C or 35°C, as in A, before the chemiluminescent signal of 3HA-Bet1 immunoprecipitated with 6myc-Sed5 was quantified by a luminoimage analyzer. The signal from the reaction at 0 °C with Sly1 was considered to be a control value. Signals from other reactions were counted and divided by the control value. The control value was subtracted from other values, and increased values of each reactions are indicated in the graph. The average of three experiments and standard error are indicated.