Figure 5.

Precipitation of active Res-FLAG in digitonin by biotinylated Sens-myc. HeLa cells (500 μg of cell protein) transfected with equal amounts of Res-FLAG and Sens-myc cDNA, or with Res-FLAG or Sens-myc alone, were treated with 1 mM MTSEA-biotin for 10 min, were washed, and were solubilized in 0.5 ml of binding buffer (18) containing 1% digitonin. The lysate was incubated with 0.5 ml of packed streptavidin-agarose beads for 2 h at 4°C, and the beads were washed three times with the same buffer containing 0.06% digitonin. Each sample of packed beads was incubated with 500 μl of 0.03 nM 2β-carbomethoxy-3β-(4-[¹²⁵I]iodophenyl)tropane ([¹²⁵I]β-CIT) in binding buffer for 30 min at 25°C in the presence (where indicated) of 100 μM cocaine, and the beads were then collected on #32 glass-fiber filters (Schleicher & Schuell), were washed, and were counted.