Gas6 and the Receptor Tyrosine Kinase Axl in Clear Cell Renal Cell Carcinoma (original) (raw)
Figure 2
Axl but not Gas6 is expressed in ccRCC 786-O cells and can be functionally active.
Expression of Axl (A) and Gas6 (B) were analyzed by western blot analysis using total cell lysate (tcl; Axl) and immunoprecipitate (Gas6) separated on an 8% reducing SDS-PAGE gel, and using Axl (Axl-C20) and Gas6 (R&D) specific antibodies, respectively. Gas6 expression in 786-O cells, grown in conditioned medium with and without vitamin K, was compared to expression in stably transfected Hek293 cells [51]. Gas6-dependent Axl phosphorylation was analysed in serum-starved (1% FCS; 20 h) 786-O cells stimulated with various Gas6 concentrations for 10 min (C) and stimulated with 400 ng/mL Gas6 for up to 1 h (D). Total cell lysate of stimulated cells were subjected to Axl immunoprecipitation with an in house Axl-specific polyclonal antibody (R042) and immune-complexes were thereafter separated on 8% reducing SDS-PAGE and immunoblotted for phosphorylated Axl (PY99). The membranes were stripped and reblotted for total amount of immunoprecipitated Axl (Axl-C20). Representative western blots are shown.