Phospho-AXL (Tyr702) Antibody - #AF8523

Figure 5 Inhibition of AXL/MERTK suppresses ICC progression and Treg numbers. (A) Experimental strategies for R428/Vehicle treatment in ICC mice. (B) Representative liver morphology image of different treatment groups for survival outcome analysis (left). Kaplan-Meier survival curve for ICC mice after R428 treatment (right). (C) Representative image of liver morphology after R428 treatment for 6 weeks (left). Statistical analysis of liver to body weight ratio after R428 treatment (right). (D) Fluorescence images of lineage tracing after R428 treatment (left). Scale bar: 50 μm. Statistical analysis of Tom+ cell number (middle) and area (right) after treatment. (E–G) Flow plots (E) and graphs of tumor-infiltrating CD4+ (F) and CD8+ (G) T cell frequency after R428 treatment. (H–J) Flow plots (H) and graphs of tumor-infiltrating NK (I) and NKT (J) cell frequency after R428 treatment. (K) Volcano plot of differentially expressed genes between R428- and vehicle-treated murine ICC Reg-TAMs, with genes meeting P < 0.01 and fold change ≥2 or ≤–2 shown in red. (L) KEGG pathway analysis of the up- and downregulated differentially expressed genes in ICC tumor cells after R428 treatment. (M) Western blot analysis of NF-κB1, AKT, p-AXL, p-MERTK, p-AKT, and GAPDH in ICC Reg-TAMs after R428 treatment. (N) qRT-PCR analysis of differentially expressed genes in ICC tumor cells after R428 treatment. (O) Dot plots showing specific expression of Ccl8 in MoMϕDC cluster (top) and Reg-TAMs (bottom). (P) ELISA showed CCL8 protein levels in ICC Reg-TAMs between R428 and vehicle treatment groups. (Q and R) Representative flow plots (Q) and Treg frequency graph (R) after R428 treatment. Data represent the mean ± SD (C, D, F, G, I, J, N, P, and R). P values were calculated by 2-tailed, unpaired Student’s t test (C, D, F, G, I, J, N, P, and R) and log-rank test (B). CTL, control.

Figure 2. Axl-S isoform has a more robust binding ability to Gas6 ligands. (A) Western-blot was used to detect the over-expression of Axl-L and Axl-S isoforms in HepG2 cells. (B) Left: Co-IP assay was used to detect the binding ability of Axl-L and Axl-S isoforms to Gas6 ligands; right: The relative amount of Gas6 protein bound in HepG2 cells over-expressing Axl-L or Axl-S. Data were analyzed using Student’s T-test. The control levels were set to a value of 1. Data are presented as mean ± S.D. (N=3). The “***” indicates “P<0.001” versus the pMXs-Axl-L group. (C) The effect of Axl-L and Axl-S over-expression on Axl and downstream AKT and ERK signaling pathway proteins was detected by Western-blot. Statistical analysis of the effects of over-expression of Axl isoforms on AKT and ERK signaling pathway proteins in HepG2 cells (D) and HCCLM3 cells (E). The relative expression of p-Axl represents the relative expression of p-Axl to Axl. The relative expression of p-AKT represents the relative expression of p-AKT to total Axl. The relative expression of p-ERK represents the relative expression of p-ERK to total ERK. The relative expression of total Axl, AKT, ERK represents the relative expression of total Axl, AKT, ERK to β-actin. Two-way ANOVA and Tukey’s post-hoc test was used to analyze the data. Data are presented as mean ± S.D. (N=3). The “*, **, ***” indicate “P<0.05, 0.01, 0.001” versus the control group, respectively. "#" represents P<0.05, "##" represents P<0.01, and "###" represents P<0.001. “#, ##, ###” indicates statistical difference between over-expressed Axl-L and Axl-S experimental groups.

Fig. 3 DCC-2036 decreases the KLF5 expression through the inhibition of AXL. A DCC-2036 inhibited the activation and expression of AXL in a dose-dependent manner. MDA-MB-231 cells, HS-578T, and 4T1 cells were treated with indicated concentrations of DCC-2036 for 48 h, and then the phosphorated and total levels of AXL were detected by WB. B Gas6 increased the protein levels of p-AXL and KLF5. MDA-MB-231 cells, HS-578T, and 4T1 cells were treated with Gas6 (200 ng/ml) for 0, 30, 60, 90, 120 min. C The knockdown of AXL reduced the protein levels of p-AXL, AXL, and KLF5. MDA-MB-231 cells were transfected with human AXL siRNA or control siRNA (NC) for 48 h, and then the protein levels were tested by WB. D The mRNA levels of AXL, KLF5, and Nanog were quantified by q RT-PCR. The statistical significance was determined by Student’s t-test, **P 

FIGURE 5 | TAM receptor expression in MAECs and Axl siRNA transfection efficiency. (A, C) Western blotting analysis of the expression of TAM receptors (Tyro3, Axl, and Mertk) in MACEs (n = 3, means ± SD). (B, D) Axl was activated when MAECs were incubated with Gas6; Western blotting was used to examine the Axl phosphorylation expression (n = 3, means ± SD, **P < 0.01 versus the control group).