Figure 3: AXL mediates EGFR nuclear translocation by enhancing YES and LYN mRNA expression.

(A) HP and Ctx^R clones were incubated with siAXL or siNT for 72 hours prior to harvesting whole-cell lysate and immunoblotting for the indicated proteins. (B) mRNA was harvested from Ctx^R clones and HP cells 72 hours post transfection with siAXL or siNT. YES and LYN mRNA expression was detected by qPCR and normalized to the expression of each target in siNT-transfected cells. β-Actin was used as an endogenous control. Data are mean ± SD of 3 in three independent experiments. ** P < 0.01 by the Mann-Whitney U test. (C) Whole cell lysate was harvested from HP and HN4 cells stably overexpressing pcDNA-AXL or Vector control and subsequently subjected to immunoblot analysis. (D) Whole cell, non-nuclear and nuclear proteins were harvested from HP and HN4 cells stimulated with Gas6 (300 ng/mL) for 30 min followed by immunoblotting for the indicated proteins. (E) HC4 cells were transfected with siAXL (50nM) or siNT for 24 hours prior to overexpression of pcDNA6.0-LYN for an additional 48 hours. Whole cell, non-nuclear, and nuclear lysate was subsequently harvested followed by immunoblotting for the indicated proteins. For presented immunoblots, GAPDH, α-Tubulin, and lamin-B were used as loading and purity controls for whole cell, non-nuclear and nuclear lysates, respectively. All immunoblots are representative of three independent experiments, and ImageJ software was used to quantify nEGFR abundance. Data in (E) are mean ± SD of three independent experiments. ** P < 0.01, by two-tailed Student t-tests.