Af885

Primary murine macrophages were generated by flushing the bone marrow from the femur and tibia of 6–8 week-old C57BL/6 mice followed by incubation for 5 days in DMEM containing 10% FBS and 10 ng/mL murine M-CSF (Peprotech). Primary pancreatic stellate cells were isolated from the pancreas of C57BL/6 mice by density gradient centrifugation, and were cultured on uncoated plastic dishes in IMDM with 10% FBS and 4 mM L-glutamine. Under these culture conditions pancreatic stellate cells activated into myofibroblasts.
To generate macrophage and fibroblast conditioned media, cells were cultured in serum free media for 24–36 h, supernatant was harvested, filtered with 0.45 μm filter, concentrated using StrataClean Resin (Agilent Technologies) and immunoblotted for Gas6 (R&D Systems, AF885).

Recombinant human Gas6 (885-GS, R & D Systems, Minneapolis, MN, USA) was used to stimulate H1299 cells. For detecting phosphorylation, cell extracts were prepared by lysing the cells in buffer containing 1 mM EDTA, 5 mM NaF, 0.5 mM sodium orthovanadate, 1 mM dithiothreitol (DTT), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and protein inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Lysates from whole cells were separated by 7.5% or 10%SDS-PAGE. Proteins were then transferred onto nylon membranes (Amersham, Buckinghamshire, UK) and incubated with mouse anti-α-SMA antibody (clone 1A4, Sigma-Aldrich), mouse anti-vimentin antibody (vim3B4, Dako), rabbit anti-PDGFR-α antibody (#3164, Cell Signaling Technology), rabbit anti-PDGFR-β antibody (#3169, Cell Signaling Technology), goat anti-Axl antibody (AF154, R&D Systems), rabbit anti-phospho Axl antibody (AF2228, R&D Systems), goat anti-Gas6 antibody (AF885, R&D Systems) or mouse anti-GAPDH antibody (Chemicon, Temecula, CA, USA) according to the manufacturers’ protocols. Proteins were detected with horseradish peroxidase-conjugated anti-rabbit, anti-goat, or anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and ECL reagents (GE Healthcare, Milwaukee, WI, USA). The blots were scanned using an imaging densitometer LAS-3000 mini (Fujifilm, Tokyo, Japan).