Figure 6
The AXL ligand, GAS6, is highly expressed in TAE684- and LDK378-resistant cells and leads to AXL activation. (a) Western blot analysis of GAS6 levels in cell lysates from parental SH-SY5Y and TAE684-resistant SY5Y-TR1 cells (left). qRT–PCR analysis of the GAS6-SV variant in the same cells (right). ***P<0.001. (b) Western blot (left) and qRT–PCR analysis (right) of the GAS6-SV variant in the parental and LDK378-resistant SH-SY5Y cells. ***P<0.001. (c) Western blot analysis of the 50 kDa cleaved GAS6 product in conditioned media from SH-SY5Y and SY5Y-TR1 cells. Arrows indicate both full-length and cleaved GAS6 forms. (d) Western blot analysis of the indicated proteins in SY5Y-TR1 cells expressing either GAS6 shRNAs or a control shRNA (scramble). Three different shRNAs (1, 2 and 3) were used. (e) Western blot analysis of total and pAXL and ERK levels, as well as GAS6 protein in SH-SY5Y cultivated for 3 days in conditioned media from control SH-SY5Y or TAE684-resistant SY5Y-TR1 cells. (f) Dose–response curves for parental SH-SY5Y cultivated for 3 days in conditioned (Cond) media from control SH-SY5Y or TAE684-resistant SY5Y-TR1 cells and then treated with TAE684 or R428 for 3 days (TAE684: IC50=SH-SY5Y Ctl Media, 66 nM; SH-SY5Y Cond Media TR1, 385 nM; R428: IC50=SH-SY5Y Ctl Media, 4085 nM; SH-SY5Y Cond Media TR1, 1836 nM).
