Anti axl c89e7 rabbit mab

Protein samples were boiled for 5 min with Laemmli sample buffer (Bio-Rad Laboratories) and loaded into pre-poured Tris-HCl-glycine SDS-PAGE gels (stacking gel 4%, resolving gel 6% or 8%). Gels run at 150 V for 1.5 hours following transfer to a polyvinylidene difluoride membrane (PVDF, Bio-Rad Laboratories) at 40 mA constant current for 2 h. Blots were blocked with 5% BSA in 1xTBST, primary and secondary antibodies were dissolved in TBST. Following primary antibodies were used. R & D Systems: human phopho-AXL (Y779) mAb (Clone 713610), Cell Signaling: anti-AXL C89E7 rabbit mAb, phospho-MET (Tyr1234/1235) rabbit mAb (D26) XP^®. Santa Cruz: Gas6 antibody (C-20). Sigma Aldrich: mouse monoclonal β-actin antibody (clone 1A4). Immunocomplexes were visualized using a second HRP-conjugated anti-rabbit or anti-mouse antibody (Pierce Biotechnology). For development we used ECL Kit (Sigma). ImageJ Software was used for densitometry analysis. Reported values were first normalized to the loading control and then multiplied by a constant to reach the lowest whole integral. Each western blot was carried out with a negative control consistent of sample diluent or beads incubated with antibody and sample diluent only.

Adherent cells were plated onto cover slips and fixed with 4% PFA for 10 minutes at room temperature. Cells were blocked with 1% casein for 45 minutes at room temperature. Antibodies were diluted in 0.5% casein. The primary antibody was incubated for one to two hours, the secondary antibody for one hour. Wash steps were carried out with TBST. Negative control was carried out without primary antibody incubation and is shown in Supplementary Figure S1 for anti-AXL antibody and anti-phospho-AXL antibody.
For immunofluorescence human phopho-AXL (Y779) mAb (Clone 713610, R & D Systems) and anti-AXL C89E7 rabbit mAb (Cell Signaling) were used. The following secondary, fluorescently labeled antibodies were used: FITC-conjugated donkey anti-rabbit IgG (Dianova, 711-095-152), Cy3-conjugated donkey anti-mouse IgG (Dianova, 115-165-146).