![Figure 1. Constitutive and inducible CX3CL1 cleavage in CX3CL1–ECV-304 cells. (A) PMA enhances the release of soluble CX3CL1 from CX3CL1–ECV-304 cells: cells were stimulated with 200 ng/μL PMA or left unstimulated for different lengths of time. Conditioned media were harvested, concentrated 10-fold, and analyzed by Western blotting using a specific antiserum against CX3CL1. (B) The broad-spectrum metalloproteinase (MP) inhibitor batimastat blocks constitutive and inducible CX3CL1 cleavage: CX3CL1–ECV-304 cells were treated with 20 μM batimastat or vehicle control (dimethylsulfoxide [DMSO]) and subsequently stimulated with 200 ng/mL PMA or left unstimulated. After 3 hours, conditioned media were harvested and analyzed for the presence of soluble CX3CL1. (C) Effect of PMA and batimastat on CX3CL1 surface expression: CX3CL1–ECV-304 cells were treated with inhibitor and stimulated with PMA as described in panel B and subsequently investigated for the surface expression of CX3CL1 by flow cytometry using a fluorescently labeled monoclonal antibody to CX3CL1. For each condition the median fluorescence intensity was calculated and given as mean and SD of triplicate determinations (values in parentheses). Unspecific antibody binding was evaluated with a fluorescently labeled IgG1 control and was not modulated by treatment with PMA or batimastat (MFI = 6 ± 1; not shown). Results shown in panels A-C are representative for 3 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/102/4/10.1182_blood-2002-12-3775/6/h81634759001.jpeg?Expires=1735503765&Signature=Z7XqElvyASjH40rAq0CyXub4~v-UErx6uJ9T5mZ24oUUwN7Xy~SFjkke1xvlaxMKjmWQCLpSumtvuz28WSXlgXJAL8BZydTLoqBERBWI9MBWw5rP5O5TqS5v9hTpHEH26xn2W1P127M6N1BcFC9pQMrSO4SoNXLmOg3PDLhAPFWP6evyu6341M-M5kZzVaqJxg-C4G3VWbT4j69zqicXaY~oyNdKrjFABiJkML73lU~elLeOlJg9xWZ818GjSdI4AKuOdm2QhRU7mzwwE8a7HmWnv~dFAEUo4S1HmxqaAjY833RfCTRpmQKubULbdNQf~sg7t~86~HeIrDnfvcxnOA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Constitutive and inducible CX3CL1 cleavage in CX3CL1–ECV-304 cells. (A) PMA enhances the release of soluble CX3CL1 from CX3CL1–ECV-304 cells: cells were stimulated with 200 ng/μL PMA or left unstimulated for different lengths of time. Conditioned media were harvested, concentrated 10-fold, and analyzed by Western blotting using a specific antiserum against CX3CL1. (B) The broad-spectrum metalloproteinase (MP) inhibitor batimastat blocks constitutive and inducible CX3CL1 cleavage: CX3CL1–ECV-304 cells were treated with 20 μM batimastat or vehicle control (dimethylsulfoxide [DMSO]) and subsequently stimulated with 200 ng/mL PMA or left unstimulated. After 3 hours, conditioned media were harvested and analyzed for the presence of soluble CX3CL1. (C) Effect of PMA and batimastat on CX3CL1 surface expression: CX3CL1–ECV-304 cells were treated with inhibitor and stimulated with PMA as described in panel B and subsequently investigated for the surface expression of CX3CL1 by flow cytometry using a fluorescently labeled monoclonal antibody to CX3CL1. For each condition the median fluorescence intensity was calculated and given as mean and SD of triplicate determinations (values in parentheses). Unspecific antibody binding was evaluated with a fluorescently labeled IgG1 control and was not modulated by treatment with PMA or batimastat (MFI = 6 ± 1; not shown). Results shown in panels A-C are representative for 3 independent experiments.
