Fig. 3. Effect of overexpressed ADAM19 on levels of mature Kitl1. (A) COS-7 cells were transfected with AP-tagged Kitl1 (top panel), or with Kitl1 together with either the catalytically inactive ADAM19E>A mutant (second panel from the top), or wild-type ADAM19 (third panel from the top), or wild-type ADAM19 in the presence of the proteasome inhibitor MG132 (lower panel) labeled with [³⁵S]-methionine for 30 minutes, and chased in OPTI-MEM medium. The cell lysates were collected at the indicated time points, immunoprecipitated with anti-Kitl antibody, and the immunoprecipitated material was separated by 8% SDS-PAGE and visualized by autoradiography. A black arrow indicates the mature form of Kitl1 (125 KDa), an asterisk is next to the intermediate form I (112.5 KDa) and a black arrowhead points to the intermediate form II (100 KDa). It should be noted that immunoprecipitated AP-tagged Kitl1 runs as a monomer on SDS-PAGE since it is reduced and boiled in SDS sample buffer. Therefore, it has a different mass than in the in-gel AP-assay shown in Fig. 1A, where AP-tagged Kitl1 runs as a dimer since the material is not boiled before SDS-PAGE. (B) Effect of ADAM19 on Kitl1 that can be biotinylated on the cell surface. Kitl1 was expressed in COS-7 cells together with either ADAM19 or ADAM19E>A, and cell surface proteins were labeled with a non-membrane-permeable biotinylation reagent as described in the Materials and Methods. The upper panel shows a western blot of Kitl1 in cell lysates, and the lower panel shows cell-surface biotinylated material that was immunoprecipitated with an antibody against the alkaline phosphatase moiety attached to Kitl1, transferred to nitrocellulose and probed with HRP-labeled streptavidin (avidin). The amount of Kitl1 that can be cell-surface biotinylated is reduced in the presence of ADAM19.