Interaction of recombinant moesin with the cytoplasmic region of PSGL-1 and ICAM-3.

(A) Coomassie-blue–stained 12% SDS-PAGE results with recombinant full-length moesin (moesin) and the amino-terminal domain of moesin (N-moesin) purified from bacteria. Small amounts of contaminating peptides are present. (B) Glutathione-Sepharose (Glut-Seph), GST, or GST-fusion proteins containing the cytoplasmic domain of PSGL-1 (CyPSGL-1) or ICAM-3 (CyICAM-3) linked to glutathione-Sepharose beads were incubated with purified recombinant full-length moesin. GST, GST-fusion proteins, and other bound proteins were eluted together from the beads with Tris buffer containing 20 mmol/L of glutathione. Eluted proteins were resolved by 10% SDS-PAGE followed by immunoblotting with 95/2 pAb specific for moesin. (C) Binding assay of the amino-terminal region of moesin (N-moesin) to the cytoplasmic tail of PSGL-1 and ICAM-3 was performed as described above (Figure 6B); proteins were separated by 10% SDS-PAGE and immunoblotted with the 95/2 pAb. To detect N-terminal moesin, a chemiluminescence system and a longer exposure than that used to obtain the results shown in Figure 6B were required because of the lower reactivity of the 95/2 pAb for the amino-terminal fragment compared with the full-length form of moesin. Molecular masses in kilodaltons are indicated on the left in Figure 6A and 6C and on the right in Figure 6B.