NE proteolytically cleaves the G-CSFR on PMN. PMN were treated with 150 μg/ml NE for the indicated times, the reactions quenched by addition of 10% FBS and 1 mM PMSF, and the cells lysed. Samples were resolved on 10% polyacrylamide gels, transferred to nitrocellulose, and the membranes immunoblotted with (A) the monoclonal antibody LMM741 recognizing FNIII domains in the extracellular portion of the G-CSFR (BD PharMingen). The arrow indicates the full length G-CSFR. (B) The blot in (A) was stripped then reblotted with rabbit polyclonal antibody recognizing the C-terminus of the G-CSFR (Santa Cruz). +FBS denotes lysates from PMN treated with NE for 120 min in the presence of 10% FBS. The arrows indicate the full length G-CSFR and resultant C-terminus generated by NE cleavage. A representative blot from three independent experiments (n = 3) is shown.