Effect of MP inhibitors on CX3CL1 cleavage in CX3CL1–ECV-304. (A-B) MP inhibitors differentially block constitutive and PMA-inducible release of the CX3CL1 ectodomain: CX3CL1–ECV-304 cells were treated with various concentrations of the natural metalloproteinase inhibitors TIMP-1 or TIMP-2 (A) or hydroxamate inhibitors (mixed ADAM10/TACE inhibitor GW280264X or selective ADAM10 inhibitor GI254023X) (B). Subsequently, cells were stimulated with 200 ng/mL PMA or left unstimulated. Generation of soluble CX3CL1 was analyzed by Western blotting of the conditioned media using a specific antiserum against CX3CL1. One representative of 3 independent experiments is shown. The CX3CL1-specific signal intensity for each lane was quantified by densitometry and expressed in relation to the control representing CX3CL1 released from unstimulated cells in the absence of inhibitor. (C) MP inhibitors differentially restore CX3CL1 surface expression: CX3CL1–ECV-304 cells were left unstimulated or stimulated with 200 ng/mL PMA in the presence and absence of mixed ADAM10/TACE inhibitor GW280264X or selective ADAM10 inhibitor GI254023X (both 1 and 3 μM). After 1 hour CX3CL1 surface expression was assayed by flow cytometry, and the CX3CL1-specific fluorescence signal was recorded as median fluorescence intensity (MFI) and calculated as mean and SD of triplicates performed in a representative of 3 independent experiments.