Fig. 1. Effect of overexpression of ADAM19 on Kitl1 shedding. (A) Wild-type ADAM19 (lanes 1 and 2) or ADAM19 carrying an inactivating E>A mutation in its catalytic site (A19 EA) (lanes 3 and 4) were transfected into COS-7 cells together with AP-tagged Kitl1 (upper panel) or TGF (lower panel). Kitl1 shedding was stimulated with 25 ng/ml PMA as described in the Materials and Methods. Briefly, medium was collected before and after addition of PMA in the same well for 1 hour and analyzed on 8% SDS-PAGE. Comparison of cell lysates expressing Kitl1 or TGF and either ADAM19 or the E>A mutant showed similar expression levels of pro-TGF and two forms of Kitl1 (black and open arrowhead), whereas much less of the slowest migrating form of Kitl1 (indicated by an asterisk) was seen in cells expressing ADAM19 compared with cells expressing the E>A mutant. (B) Western-blot analysis of the expression of Kitl1 (upper panel) and ADAM19 or ADAM19 E>A (lower panel) in COS-7 cells. The slowest migrating form of Kitl1, which is not present in the sample overexpressing ADAM19, is indicated by an asterisk.