Fig. 1
The novel monoclonal antibody anti-M7 selectively blocks the CD40L-binding site within the Mac-1 (CD11b/CD18) I-domain. a The peptide sequence M7 within the Mac-1 I-domain, required for binding of CD40L, is a highly conserved binding motif between the human and mouse integrin. The antibody anti-M7 was generated by immunization of mice with the binding peptide VMEQLKKAKTLMQ coupled to diphtheria toxoid. b Anti-M7 bound to a CHO cell line over-expressing native human (Mac-1 WT) and permanently activated human Mac-1 (Mac-1 del), but not to control CHO cells that did not express Mac-1 (CHO) in western blot. c Specific binding of the antibody anti-M7 to the immobilized peptides M7 (EQLKKSKTL), the scrambled control sM7 (KLSLEKQTK), and the closely located peptide M8 (EEFRIHFT) in a solid-phase binding assay with immobilized peptides. d Anti-M7 was coupled with the fluorochrome Alexa-647 and binding to human leukocyte subsets was quantified in flow cytometry. An Alexa-647 labeled IgG isotype antibody served as control. e, f Adhesion of CHO cells over-expressing the permanently activated Mac-1 mutant (del) on dishes coated with immobilized human CD40L or alternative human Mac-1 ligands in a static adhesion assay. Cells were incubated with anti-M7 or the human pan I-Domain blocking anti-human reference anti-Mac-1 (clone 2LPM19c) 15 min prior to adhesion. g CHO-Mac-1 cell adhesion on immobilized CD40L was blocked with Fab fragment preparations of IgG, anti-M7 or anti-human Mac-1 (2LPM19c). Error bars indicate mean ± SEM. Statistical significance was assessed by an unpaired, two-sided Student’s T-test between the indicated groups, **P < 0.01, ***P < 0.001, ****P < 0.0001 (f, g) or against IgG if not otherwise indicated (f). Data are the result of N ≥ 3 independent experiments. Representative pictures are shown in b, e. res. indicates the location/residue within the Mac-1 protein sequence. Scale bar (e) represents 100 μm
