Journal:

Article Title: Dexamethasone enhances interaction of endogenous Annexin 1 with L-selectin and triggers shedding of L-selectin in the monocytic cell line U-937

doi: 10.1038/sj.bjp.0705413

Figure Lengend Snippet: L-selectin binding to ANXA1 is calcium-dependent and involves the N-terminal domain of ANXA1. (a) Comparison of the amounts of ANXA1 detected after presentation of the protein by a specific mAb or after direct binding to plastic. A monoclonal ANXA1 antibody was coated onto microtiter plates (3 μg ml−1) to capture varying concentrations of ANXA1 (mAB1-A+ANXA1). This binding was compared to the binding for the same range of ANXA1 recombinant protein concentrations directly coated on extra-high binding capacity plastic (ANXA1). In both cases, ANXA1 was detected with a polyclonal ANXA1 antibody and showed no major difference between the two methods, (b–d) Direct binding between annexins and L-selectin chimeric protein (Lec-IgM) in the presence or absence of calcium, (b) Increasing concentrations of Lec-IgM (black square) were immobilized on plastic and then incubated with 9.4 μg ml−1 of ANXA1. Internal control for nonspecific binding was determined in the presence of human IgM (hIgM) (open square), (c) Lec-IgM or human IgM were added to increasing concentrations of ANXA1, either directly coated on the plastic (ANXA1) or presented by the monoclonal antibody mAB1-A (mAB-1A+ANXA1). Presence of the ANXA1 in the assay was detected with a polyclonal antibody (mAB1-A+ANXA1) as in (a), (d) Effect of the presence of calcium of the N-terminal domain of ANXA1 on the interaction between ANXA1 and Lec-IgM. Binding of Lec-IgM (0.125 μg ml−1) to increasing concentrations of ANXA1 was compared to the binding to the same concentration range of the chimeric protein ANXA1-5 (N-terminal domain of ANXA1 (1–26) and core domain of ANXA5) or to an unrelated protein, ANXA5. Experiments were done in PBS+1 mM Ca2++1 mM Mg2+ with or without 10 mM EDTA (+EDTA). Each point represents the mean±s.e.m. of three independent experiments done in duplicates.

Article Snippet: Measurement of L-selectin binding to immobilized annexin proteins The sandwich ELISA used to determine L-selectin binding to ANXA1 was performed in 96-well microtiter plates with extra-high binding capacity for proteins (Immulon 4HBX, Dynex, Chantilly, VA, U.S.A.).

Techniques: Binding Assay, Comparison, Recombinant, Incubation, Control, Concentration Assay