Surface expression and iC3b binding of WT and mutant CD11b/CD18 in HEK293 cells. (A) Histograms showing the relative binding of the heterodimer-specific mAb IB4 and the anti-CD11b mAb 44a to HEK293 cells expressing WT and βTD mutant CD11b/CD18 receptors. Binding of the respective antibody to the WT receptor was considered 100. Each histogram represents mean ± SD of triplicate determinations from a representative experiment (1 of 3 performed). (B) Western blots following 4% to 15% gradient SDS-PAGE showing the presence of CD18 in anti-CD11b (using mAb 44a) immunoprecipitates from HEK293 cells expressing WT (lane 1), AGAA (lane 2), DSSG (lane 3), NGTD CD11b/CD18 mutants (lane 4), and mock-transfected cells (lane 5). Approximately equivalent amounts of the immunoprecipitated receptors were loaded in each lane. The shift in CD18 in the NGTD mutant (lane 4) is consistent with attachment of a neo–N-glycan. Arrowheads indicate molecular weight markers at 100 kDa and 75 kDa (High MW Markers; Boston Bioproducts). No CD18 was seen in anti-CD11b immunoprecipitates from mock-transfected HEK293 cells (lane 5). (C) Histograms showing the relative binding of EiC3b to WT and βTD mutant CD11b/CD18 in buffer containing EDTA (5 mM), Ca²⁺ and Mg²⁺ (1 mM each), or Mn²⁺ (1 mM) and expressed as percentage of binding to WT obtained in the presence of 1 mM each of Ca²⁺ and Mg²⁺ after correcting for expression using mAb IB4 as described.⁹  Similar results were obtained when the activation-insensitive mAb 44a was used as reference (not shown). Each bar represents mean ± SD of triplicate determinations from a representative experiment (1 of 3 performed).