FIG. 2.

Gas6 activates Axl RTK in C57MG mammary cells. (A) Analysis of Axl and Rse RTK protein expression and regulation during the assessment of density growth arrest. C57MG cells were seeded as described for Fig. 1A, and the cellular lysates were prepared after 1, 2, 3, and 4 days by adding Laemmli loading buffer directly to the petri dish. Equal amounts of total proteins, as determined using the Bio-Rad assay, were loaded on SDS-PAGE gels and were blotted to nitrocellulose membranes. Western blots were decorated separately by using rabbit affinity-purified polyclonal antibodies for Axl, Gas1, and p27; a goat polyclonal antibody for Rse; or a monoclonal antibody for tubulin. The complexes were evidenced by incubation with a secondary peroxidase-conjugated antibody and the ECL method. (B) Gas6 activates Axl RTK. Western blot analysis with antiphosphotyrosine antibodies of Axl and Rse RTKs immunoprecipitated (IP) from density-inhibited C57MG cells. Both antibodies recognizing the C-terminal region (upper panel) and the N-terminal region of Axl and Rse RTKs were used in this analysis. The results shown here are representative of several experiments. −, absence of Gas6; +, presence of Gas6.