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. 2016 Dec 29;31(4):1382–1397. doi: 10.1096/fj.201600702R

Figure 5.

Figure 5.

Abolishment of the kinase activity of AXL accelerates its proteolysis by α-secretase. A) AXL kinase activity is not required for the regulated proteolysis of AXL. HEK293T cells were transiently transfected with AXL-FL WT or K567R mutant for 24 h and further incubated with 10 μM DAPT before analysis by Western blot of AXL-FL and -CTF protein levels (anti-Flag; left). AXL-CTF levels of DAPT-treated cells transfected with AXL-FL WT or K567R were quantified and expressed as a percentage of WT values (right). Data are shown as means ± sem (n = 4). ***P < 0.001, unpaired Student’s t test. B) The processing of AXL is increased by an AXL kinase inhibitor. Panc-28 cells were incubated overnight with DMSO or 150 nM R428, a selective AXL kinase inhibitor, before examining by Western blot the levels of endogenous AXL-FL and AXL-CTF (C-20; left). Levels of AXL-CTF in DAPT-treated cells were quantified as described above and unpaired Student’s t test was employed for the analysis (right). Data are shown as means ± sem (n = 4). ***P < 0.001. C) Phosphorylated or nonphosphorylated AXL-CTF is processed with the same potency by γ-secretase. HEK293T cells expressing WT AXL-FL or the mutant AXL-FL K567R mutant were lysed and equal amounts of whole-cell lysates (WCL) were precipitated with the disuccinimidyl suberate–cross-linked AXL (C-20) Ab. Eluted proteins were then resolved by PAGE on a 10% Tris-glycine SDS gel and blotted with anti-pY (4G10) and anti-Flag Ab for phosphorylated (top) and total (middle) AXL-FL or AXL-CTF, respectively. Relative levels of phosphorylated AXL-CTF [the anti-pY blot for AXL-wild-type (WT AXL-FL); top] and nonphosphorylated AXL-CTF (the anti-Flag blot for AXL-K567R mutant; middle) in the immunoprecipitated (IP) samples were quantified by densitometry and expressed as percentage of DMSO controls and shown as means ± sem (n = 3). Relative levels of AXL-CTFs in the DMSO groups from the anti-Flag blot (middle) were additionally displayed (AXL-WT, 100%; means ± sem; n = 3). *P < 0.05, paired Student’s t test. Immunoblots show representative results of at least 3 independent experiments. β-Actin or GADPH serves as the loading control.