Journal: Molecular and Cellular Biology
Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum
doi: 10.1128/mcb.25.21.9324-9339.2005
Figure Lengend Snippet: FIG. 8. Immobilized Axl-Fc chimeric protein promotes cell migra- tion and induces the phosphorylation of Axl and PI3K. (A) L929R cells were plated onto Axl-Fc- or IL-3R–Fc (control)-coated six-well plates and allowed to grow in the presence of 10% FCS. After 18 h, a wound was created by scratching with a pipette tip (0 h). The cells were washed with PBS and incubated further to allow migration into the wounded area. Phase-contrast images of matched pairs of marked wound regions were taken 6 and 18 h later to assess cell migration. (B) L929R cells were serum starved for 4 h and incubated in Axl-Fc- coated wells for 15 min. Incubation in IL-3R–Fc (control)-coated wells or stimulation with 100 ng/ml of Gas6 was used as a negative or positive control, respectively. Cells were lysed, and Axl or PI3K was immunoprecipitated from the lysates with specific Abs. Precipitates were subjected to 10% SDS-PAGE and analyzed with anti-pTyr Abs. Detection of Axl or PI3K on the same blots was used as a loading control. The picture is representative of three independent experi- ments, all of which yielded highly comparable results. IP, immunopre- cipitation; WB, Western blotting.
Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.
Techniques: Phospho-proteomics, Control, Transferring, Incubation, Migration, Positive Control, Immunoprecipitation, SDS Page, Western Blot