Constitutive CX3CL1 cleavage is reduced in murine embryonic fibroblasts (MEFs) lacking ADAM10. (A) Deficiency of ADAM10 protein in MEFs with targeted disruption of the Adam10 gene: SDS lysates of Adam10^+/+ or Adam10^–/– MEF cell lines were analyzed for the presence and absence of ADAM10 by Western blotting using an antiserum against murine ADAM10. (B) CX3CL1 shedding in Adam10^+/+ and Adam10^–/– MEF cell lines: CX3CL1 was transiently transfected into Adam10^+/+ or Adam10^–/– MEFs. Cells were stimulated with 200 ng/mL PMA for 1 hour, and subsequently conditioned media and cell lysates were analyzed for the presence of soluble and cell-associated CX3CL1, respectively, by ELISA. To take account of variations in the transfection efficiency, for each single transfection data were calculated as the percentage of soluble CX3CL1 released in the medium in relation to the total amount of soluble and cellbound CX3CL1 determined in the medium and the lysate. Results are shown as mean and SD of triplicate transfections of one experiment. In Adam10^–/– MEFs, shedding was significantly reduced compared with that observed in Adam10^+/+ MEFs (P < .05, indicated by asterisks). The increase in shedding due to PMA stimulation (determined as difference in shedding between unstimulated and PMA-stimulated cells) was similar in both cell lines (P < .05, not indicated). In panels A-C, 1 representative of 3 independent experiments is shown.