Biotinylation of c-kit protein in the absence or presence of SLF.

MO7e cells were treated with or without 50 ng/mL SLF for 90 minutes at 37°C. Then, cell surface proteins were labeled with biotin. The cells were solubilized with lysis buffer containing 1% CHAPS. Cell lysates were immunoprecipitated with indicated first antibodies. Immunoprecipitated materials were eluted with 1% NP-40 lysis buffer and supernatants were immunoprecipitated with anti–c-kit polyclonal antibody. Immunoprecipitated proteins were separated by SDS-PAGE and transferred onto Immobilon-P membrane. Biotin-labeled proteins were revealed with streptavidin-biotinylated horseradish peroxidase complex. These are the representative results from 2 separate experiments.