P axl

Manufactured by Cell Signaling Technology

Sourced in United States

P-AXL is a phospho-specific antibody that recognizes the phosphorylated form of the Receptor Tyrosine Kinase AXL. It is used to detect and quantify the activation state of AXL in cellular samples.

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Close spelling variants of this product

P‐AXL(Tyr702) (4 citations) Anti-p-AXL (Y702) (2 citations)

14 protocols using p axl

Comprehensive Protein Expression Analysis

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Whole-cell lysates were prepared as previously described^{55 (link)}. Antibodies specific for p53 (1:2000, sc-126), p-EGFR (1:1000, Tyr1173, sc-101668), EGFR (1:2000, sc-373749), p-Erk (1:1000, Thr202/Tyr204, sc-16982), Erk (1:3000, sc-94), Akt (1:3000, sc-5298), AXL (1:1000, sc-1096), E-cadherin (1:1000, sc-71008), EpCAM (1:1000, sc-71059), desmoplakin (1:1000, sc-390975), cytokeratin-8/18 (1:1000, sc-70939) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); those for p-Akt (1:1000, Ser473, #4060), β-catenin (1:1000, #8480), p-AXL (1:1000, #4060), and vimentin (1:1000, #5741) were obtained from Cell Signaling Technology (Beverly, MA, USA). To assess the level of p-AXL, lysates were immunoprecipitated with an anti-AXL antibody and immunoblotted with an anti-phosphotyrosine (p-Tyr, 1:1000, sc-7020, Santa Cruz) antibody. The immunoblotting is representative of three independent experiments.

Jung S., Kim D.H., Choi Y.J., Kim S.Y., Park H., Lee H., Choi C.M., Sung Y.H., Lee J.C, & Rho J.K. (2021). Contribution of p53 in sensitivity to EGFR tyrosine kinase inhibitors in non-small cell lung cancer. Scientific Reports, 11, 19667.

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Inhibition of AXL and EGFR Signaling

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Gefitinib (CAS No. HY-50895) and Osimertinib (AZD-9291, CAS No. HY-15772) were purchased from MedChemExpress (NJ, USA). Cycloheximide (CAS No. 66-81-9) was purchased from A.G. Scientific (CA, USA). Yuanhuadine (YD; purity >98.5%) was isolated from a CHCl₃-soluble fraction of the flowers of Daphne genkwa, as described previously^{22 (link)}. All chemicals were dissolved in DMSO for in vitro experiments.
Antibodies against C-terminal AXL (sc-1096), EGFR (sc-03), p-ERK (sc-7383), ERK (sc-94), MET (sc-10), β-actin (sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-AXL (#5724), p-EGFR (#2234), p-MET (#3077), p-Akt (#9271), Akt (#9272), p-p70S6 Kinase (#9205), p70S6 Kinase (#9202), p-SAPK/JNK (#9091), SAPK/JNK (#9252), and snail (#3879) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Kim D., Bach D.H., Fan Y.H., Luu T.T., Hong J.Y., Park H.J, & Lee S.K. (2019). AXL degradation in combination with EGFR-TKI can delay and overcome acquired resistance in human non-small cell lung cancer cells. Cell Death & Disease, 10(5), 361.

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Western Blot Analysis of AXL and AKT

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Protein lysates were prepared from cultured cells or frozen tumors using a 9M Urea, 0.075 M Tris buffer (pH 7.6) and quantified using the Bradford assay. 50-100μg of protein was subjected to reducing SDS/PAGE by standard methods. Western blots were incubated with primary antibodies against AXL (R&D Systems), P-AXL (Cell Signaling), GAS-6 (R&D), AKT (Cell Signaling), and phospho-AKT (Cell Signaling) as needed. To confirm equal protein loading, blots were probed with antibodies specific for β-actin (Sigma Aldrich), HSP70 (Thermo Fisher), or Vinculin (BD Pharmingen).

Divine L.M., Nguyen M.R., Meller E., Desai R.A., Arif B., Rankin E.B., Bligard K.H., Meyerson C., Hagemann I.S., Massad M., Thaker P.H., Hagemann A.R., McCourt C.K., Powell M.A., Mutch D.G, & Fuh K.C. (2016). AXL modulates extracellular matrix protein expression and is essential for invasion and metastasis in endometrial cancer. Oncotarget, 7(47), 77291-77305.

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Comparative Analysis of EGFR Inhibitors

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Gefitinib, erlotinib, lapatinib and R428 were purchased from Selleck Chemicals (Houston, TX, USA). Propidium iodide and RNase A were purchased from Sigma-Aldrich (St Louis, MO, USA). The primary antibodies used were as follows: E1A (ab-52523; Abcam), EGFR (sc-03; Santa Cruz Technology), AXL (#4566; Cell Signaling Technology), p-AXL (#5724; Cell Signaling Technology), and α-Tubulin (T-5168; Sigma). All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

Su C.M., Chang T.Y., Hsu H.P., Lai H.H., Li J.N., Lyu Y.J., Kuo K.T., Huang M.T., Su J.L, & Chen P.S. (2016). A novel application of E1A in combination therapy with EGFR-TKI treatment in breast cancer. Oncotarget, 7(39), 63924-63936.

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Western Blot Analysis of PI3K/Akt/mTOR Pathway

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Cells were lysed in lysis buffer (Thermo Fisher Scientific, Inc.) containing phosphatase inhibitors (Thermo Fisher Scientific Inc.) and PMSF (Thermo Fisher Scientific Inc.). The proteins were loaded on an 10% SDS gel, resolved using SDS-PAGE, and transferred to PVDF membranes (MilliporeSigma). Subsequently, the membranes were blocked with 5% skimmed milk, incubated with primary antibodies against pPI3K (cat. no. ab182651; 1:200; Abcam), PI3K (cat. no. ab191606; 1:1,000; Abcam), pAxl (cat. no. 96453; 1:1,000; Cell Signaling Technology, Inc.), Axl (cat. no. ab215205; 1:1,000; Abcam), pAkt (cat. no. ab81283; 1:2,000; Abcam), Akt (cat. no. ab38499; 1:1,000; Abcam), mTOR (cat. no. ab2732; 1:2,000; Abcam), PTEN (cat. no. ab32199; 1:5,000; Abcam), or GAPDH (cat. no. ab8245; 1:2,000; Abcam), followed by incubation with the relevant horseradish peroxidase-conjugated secondary antibody (anti-Rabbit; cat. no. ab205718; anti-Mouse: cat. no. ab6789; Abcam). GAPDH was used as the internal control. Signals were visualized using a Laser Holographic imager (Azure Biosystems).

Ji J., Ding Y., Kong Y., Fang M., Yu X., Lai X, & Gu Q. (2023). Triple‑negative breast cancer cells that survive ionizing radiation exhibit an Axl‑dependent aggressive radioresistant phenotype. Experimental and Therapeutic Medicine, 26(3), 448.

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Comprehensive Reagent and Antibody Sources

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Reagents were obtained from the following sources: ADI-PEG20 (specific activity, 5∼10 IU/mg) from Polaris Pharmaceuticals Inc. (San Diego, CA); sulforhodamine B (SRB), Ly294002 from Sigma–Aldrich (St. Louis, MO); perifosine, PLX4720 and foretinib (XL880 or GSK1363089) from Selleck Lab (Houston, TX); PI-103 from Echelon Biosciences (Salt Lake City, UT); gefitinib and lapatinib from LC laboratories (Woburn. MA); JQ1 from AdooQ Bioscience (Irvine, CA).
Antibodies were obtained from the following sources: rabbit antibodies for hEGFR, p-EGFR, p-Axl, EphA2(ser897), and EphA2(Y588) from Cell Signaling (Danvers, MA); rabbit anti-Axl, p-Axl, EGFR, P-EGFR, hErbB2, p-hErbB2, HGF-1R, hIGF, EphA1, and α-tubulin antibodies were from R&D Laboratories (Minneapolis, MN). Polyclonal antiphosphotyrosine (p-Tyr) antibody from Thermo Fisher (Grand Island, NY); rabbit anti-EphA2 antibody from Bethyl laboratory (Houston, TX); anti-ASS1 antibody from Polaries.
Small interfering RNA (siRNA) for c-Myc and AXL has been published [20 (link)]; EphA2 (Cat. No. SASI_Hs01-00222676 and SASI-Hs01-000265), RSK (SASI_Hs01_00070213 and SASI_Hs02_00305126) were all obtained from Sigma-Aldrich (St. Louis, MD).

Kuo M.T., Long Y., Tsai W.B., Li Y.Y., Chen H.H., Feun L.G, & Savaraj N. (2019). Collaboration Between RSK-EphA2 and Gas6-Axl RTK Signaling in Arginine Starvation Response That Confers Resistance to EGFR Inhibitors. Translational Oncology, 13(2), 355-364.

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Protein Expression and Phosphorylation Analysis

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Cell lysates or conditioned media that was concentrated using Amicon Ultra 0.5 10K Centrifugal Filters (EMD Millipore, Billerica, MA, USA), were prepared using standard protocols. The following antibodies were used: AXL (#4566), pAXL (#5724), ALK (#3333), pALK (#3341), AKT (#4691), pAKT (#9271), ERK (#4695), pERK (#4377), VIM (#5741), CDH1 (#3195), tubulin (#2128) and actin (#4967) from Cell Signaling Technology (Danvers, MA, USA); GAS6 (sc-376087), HSP90 (sc-59577) and phosphotyrosine (sc-81529) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Flag (#F3165) from Sigma-Aldrich (Saint Louis, MO, USA); and AXL antibody for immunocytochemistry from R&D Systems (Minneapolis, MN, USA, #AF154).

Debruyne D.N., Bhatnagar N., Sharma B., Luther W., Moore N.F., Cheung N.K., Gray N.S, & George R.E. (2015). ALK inhibitor resistance in ALKF1174L-driven neuroblastoma is associated with AXL activation and induction of EMT. Oncogene, 35(28), 3681-3691.

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Extraction and Analysis of Protein Signaling

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Total protein was extracted with SDS lysis buffer (Beyotime Biotechnology). Nuclear and cytoplasmic proteins were prepared as described previously.¹⁵ Cells for IP were lysed with RIPA lysis buffer (Beyotime Biotechnology). Protein A/G Magnetic Beads were purchased from Bimake. Antibodies are listed as follows: YAP (#14074), p‐YAP (Ser127) (#13008), AXL (#8661), p‐AXL (Tyr702) (#5724), STAT3 (#9139), p‐STAT3 (Tyr705) (#9145), AKT (#4691), p‐AKT (Ser473) (#4060), ERK (#4695), p‐ERK1/2 (Thr202/Tyr204) (#4370), MMP2 (#40994), MMP9 (#13667), MST1 (#3682), p‐MST1/2 (Thr183/Thr180) (#49332), LATS1 (#3477), p‐LATS1 (Thr1079) (#8654), LATS2 (#5888), MOB1 (#13730), p‐MOB1 (Thr35) (#8699) (Cell Signaling Technology, CST), MST2 (12097‐1‐AP) (Proteintech).

Li J., Shi C., Zhou R., Han Y., Xu S., Ma H, & Zhang Z. (2020). The crosstalk between AXL and YAP promotes tumor progression through STAT3 activation in head and neck squamous cell carcinoma. Cancer Science, 111(9), 3222-3235.

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Quantifying Protein Phosphorylation Signaling

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Western blotting was performed as described previously⁸ (link) using primary antibodies for p-AXL (Tyr702), t-AXL, p-EGFR (Tyr1086), β-actin (13E5), p-Akt (Ser473), t-Akt, p-Erk1/2 (Thr202/Tyr204), t-ERK1/2 (1:1000; Cell Signaling Technology), and t-EGFR (1:1000; R&D Systems).

Nakamura R., Fujii H., Yamada T., Matsui Y., Yaoi T., Honda M., Tanaka N., Miyagawa-Hayashino A., Yoshimura A., Morimoto K., Iwasaku M., Tokuda S., Kim Y.H., Konishi E., Itoh K, & Takayama K. (2023). Analysis of Tumor Heterogeneity Through AXL Activation in Primary Resistance to EGFR Tyrosine Kinase Inhibitors. JTO Clinical and Research Reports, 4(6), 100525.

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Liver Histochemistry and Immunohistochemistry

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Liver sections were stained with hematoxylin and eosin. Immunohistochemical staining for 4-Hydroxynonenal (4-HNE) (R&D Systems) and phosphorylation AXL (p-AXL) (Cell Signaling Technology) was performed on the paraffin sections.

Zhan M., Liu D., Yao L., Wang W., Zhang R., Xu Y., Wang Z., Yan Q., Fang Q., Du J, & Chen L. (2024). Gas6/AXL Alleviates Hepatic Ischemia/Reperfusion Injury by Inhibiting Ferroptosis via the PI3K/AKT Pathway. Transplantation.

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