CD9 EC2 peptides 5b (¹³⁵K-V¹⁷²) and 6a (¹⁶⁸P-I¹⁸⁵) competitively block anti-CD9 mAb7 binding to soluble and cell surface CD9 as well as reverse the CD9 inhibitory influence on CHO cell adhesion to fibronectin.

(A) Platelet lysate was added to either anti-CD9 mAb7, control mouse IgG₁, κ (MOPC 21), or mAb7 incubated with either peptides 5a (¹¹¹Y-T¹³⁴), 5 (¹²⁵Y-I¹⁴⁶), 5b (¹³⁵K-V¹⁷²), or 6a (¹⁶⁸P-I¹⁸⁵). The immunoprecipitates (IPTs) were fractionated by SDS-PAGE and transferred to PVDF membrane. CD9 was detected using mAb7. Peptides 5b and 6a block immunoprecipitation of CD9 by antibody mAb7 from human platelet lysate. (B) The peptides 5a (¹¹¹Y-T¹³⁴), 5 (¹²⁵Y-I¹⁴⁶), 5b (¹³⁵K-V¹⁷²), or 6a (¹⁶⁸P-I¹⁸⁵) were incubated for 30 minutes in the presence of anti-CD9 antibody mAb7. A species-specific IgG and antibody mAb7 alone were used as the negative and positive controls, respectively. Flow cytometry analysis revealed peptides 5b and 6a blocked mAb7 binding as indicated by the left shift in fluorescence intensity. (C) MOCK and A6 CHO cells were allowed to adhere to FN in the presence of peptides corresponding to segments of CD9 EC2 as described in “Materials and methods.” After stringent washing, adherent cells were counted in 5 high-power (× 40) fields of view/well from 3 wells per assay and reported as the number of adherent cells/mm². Data are expressed as the means ± SEs of 3 independent assays. The presence of peptides 5b and 6a reversed the inhibitory influence of CD9 on A6 CHO cell adhesion to fibronectin.