FIGURE 5.

In vitro pulldown analysis of the ESP1-V2Rp5 interaction. A, specific interaction of ESP1 with the extracellular region of V2Rp5. Agarose beads covalently attached to ESP1 were incubated with V2Rp5 consisting of the extracellular region and a FLAG tag. After washing with 100 mm NaCl (lane 2), the bound protein was eluted with free ESP1 (lane 3) or ESP4 (lane 4). Aliquots of each fraction were subjected to a 10% SDS-PAGE and immunoblotting with an anti-FLAG antibody. B, charge-dependent interaction between ESP1 and V2Rp5. Agarose beads covalently attached to ESP1 (lanes 1–3) or ESP4 (lanes 4–6) and unattached beads (—) (lanes 7–9) were incubated with V2Rp5 then washed with buffer containing 100 mm NaCl (lanes 2, 5, and 8). Bound protein was eluted with 500 mm NaCl (lanes 3, 6, and 9). All fractions were analyzed as described in A.