mhm24 507  (Developmental Studies Hybridoma Bank)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Autonomous conformational regulation of β 3 integrin and the conformation-dependent property of HPA-1a alloantibodies

doi: 10.1073/pnas.1806205115

Figure Lengend Snippet: Conformation and ligand binding of the full-length cβ3 integrin on the cell surface detected by flow cytometry. (A and B) Binding of conformation-specific anti-β3 mAb LIBS-1 or 319.4 to HEK293FT cells transiently transfected with αIIb plus β3 or cβ3 alone. For the αIIbβ3 transfection, mAb binding was also performed in the presence of ligand-mimetic drug eptifibatide (Ept). Integrin expression was reported with the conformation-independent mAb AP3. (C) Quantitation of the conformation-specific mAb binding presented as a percentage of AP3 binding. (D) ICAM-1 binding in buffer containing 1 mM Mg2+ to HEK293FT cells transiently transfected with cβ3 WT or the metal ion-binding defective mutant cβ3-SSAA. (E) Quantitation of the ICAM-1 binding in 1 mM Mg2+ with or without the αL-specific inhibitor presented as a percentage of AP3 binding. (F and G) Expression and ligand binding of cβ3 in stably transfected CHO-k1 or HEK293 cells. The cβ3 expression was detected by the anti-β3 mAbs AP3 and SZ21 or the anti–αL-I domain mAb MHM24. Human ICAM-1 binding was measured in 1 mM Mg2+ (Mg) or 2 mM Mn2+ (Mn) with or without the αL-specific inhibitor.

Article Snippet: Integrin cell surface expression was measured by flow cytometry using the anti-β 3 mAb AP3 ( 73 ) or SZ21 (Santa Cruz Biotechnology), anti-α IIb mAb 10E5 ( 74 , 75 ), the anti-α V β 3 complex-specific mAb LM609 (EMD Millipore), and the anti-α L I domain mAb MHM24 (Developmental Studies Hybridoma Bank).

Techniques: Ligand Binding Assay, Flow Cytometry, Binding Assay, Transfection, Expressing, Quantitation Assay, Mutagenesis, Stable Transfection