Figure 3.

Endogenous AXL in cancer cells is sequentially processed by α- and γ-secretases. A–C) NSCLC cell lines (H1299 and A549; A), pancreatic cancer cell lines (AsPC-1, Panc-1, and Panc-28; B), and glioma cell lines (LN-18 and LN-229; C) were treated overnight with DMSO, 100 ng/ml PMA, 20 μM TAPI-1, 5 μM C3, or 200 nM Epo in the presence or absence of 10 μM DAPT. Cell lysates were collected for detection of AXL-FL, -CTF, or -ICD using the C-20 Ab targeting the C terminus of AXL. D, E) α-Secretases ADAM10 and TACE are in charge of the production of AXL-CTF in Panc-28 (D) and HEK293T (E) cells. Panc-28 and HEK293T cells were transfected with the indicated siRNA in the absence and presence of Flag-tagged AXL-FL, respectively, before the 16-h incubation with DAPT (for details, see Materials and Methods). Cells were then lysed to analyze AXL-FL and AXL-CTF by Western blotting using an anti-AXL Ab (B-2) and an anti-Flag Ab for Panc-28 and HEK293T cells, respectively. AXL-CTF levels in DAPT-treated groups were quantified and expressed as the percentage of control siRNA (siCTR). Data are presented as means ± sem (n = 3). Knockdown efficiencies of siRNAs for ADAM10, TACE, and BACE were validated by quantitative RT-PCR and/or Western blots in these samples, and data are shown in Supplemental Fig. 1. GAPDH and β-actin were used as equal loading controls. Asterisk indicates nonspecific band. All blots were performed at least twice, and a representative experiment is shown. *P < 0.05, ***P < 0.01, ***P < 0.001, 1-way ANOVA with Bonferroni's multiple comparisons test.