anti-phospho-axl (p-axl)  (Cell Signaling Technology Inc)

Journal: Experimental and Therapeutic Medicine

Article Title: Repurposing of the Syk inhibitor fostamatinib using a machine learning algorithm

doi: 10.3892/etm.2025.12860

Figure Lengend Snippet: List of drugs with connectivity scores >40 after knock-down of TAM kinases.

Article Snippet: Antibodies for phospho-AXL (#5724), AXL (#8661), and TYRO3 (#5585) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown

Journal: Experimental and Therapeutic Medicine

Article Title: Repurposing of the Syk inhibitor fostamatinib using a machine learning algorithm

doi: 10.3892/etm.2025.12860

Figure Lengend Snippet: Workflow of the current study. The workflow mainly consists of a computational approach and experimental validation. A total of 31 Food and Drug Administration-approved drugs with an IC 50 <1 µM in AXL, MERTK and TYRO3 were selected using the Deargen DTI model (MT-DTI). Next, transcriptome pattern analysis using the Connective Map database was performed. Fostamatinib, which was selected as the final candidate for drug repositioning, was experimentally validated. MT-DTI, molecule transformer-drug-target interaction; DB, database.

Article Snippet: Antibodies for phospho-AXL (#5724), AXL (#8661), and TYRO3 (#5585) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Biomarker Discovery

Journal: Experimental and Therapeutic Medicine

Article Title: Repurposing of the Syk inhibitor fostamatinib using a machine learning algorithm

doi: 10.3892/etm.2025.12860

Figure Lengend Snippet: Fostamatinib suppresses cell proliferation via inhibition of TAM kinases. (A) Cell viability assay of H1299, SCC4 and MB231 cells after BMS-777607 treatment for 48 h; (B) Western blot of p-AXL, AXL, p-MERTK, MERTK and TYRO3 in BMS-777607-treated cells (α-tubulin was used as a loading control); (C) Cell viability assay of H1299, SCC4 and MB231 cells after fostamatinib treatment for 48 h; (D) Western blot of p-AXL, AXL, p-MERTK, MERTK and TYRO3 in fostamatinib-treated cells (α-tubulin was used as a loading control). Data are representative of three independent experiments and are presented as the mean ± SD. The statistical significance was analyzed via Student's t-test; * P<0.05 vs. untreated group. TAM, TYRO3, AXL, MERTK; p-, phosphorylated.

Article Snippet: Antibodies for phospho-AXL (#5724), AXL (#8661), and TYRO3 (#5585) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Viability Assay, Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Repurposing of the Syk inhibitor fostamatinib using a machine learning algorithm

doi: 10.3892/etm.2025.12860

Figure Lengend Snippet: TAK-659, a Syk inhibitor, does not affect TAM signaling. (A) Cell viability assay of H1299, SCC4 and MB231 cells after TAK-659 treatment for 48 h; (B) Western blot of p-AXL, AXL, p-MERTK, MERTK, and TYRO3 in TAK-659-treated cells (α-tubulin was used as a loading control). Data are representative of three independent experiments and presented as the mean ± SD. The statistical significance was analyzed via Student's t-test. * P<0.05 vs. untreated group. TAM, TYRO3, AXL, MERTK; p-, phosphorylated; Syk, spleen tyrosine kinase.

Article Snippet: Antibodies for phospho-AXL (#5724), AXL (#8661), and TYRO3 (#5585) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Viability Assay, Western Blot, Control

Journal: Genes & Development

Article Title: Fatty acid uptake activates an AXL–CAV1–β-catenin axis to drive melanoma progression

doi: 10.1101/gad.351985.124

Figure Lengend Snippet: Reagents used in this study

Article Snippet: Rabbit polyclonal anti-phospho-Axl (Tyr779) , Cell Signaling Technology , 96453 , .

Techniques: Software, Transduction, Virus, Subcloning, Bacteria, Recombinant, Bradford Assay, Protease Inhibitor, RNA Extraction, Reporter Assay, Bicinchoninic Acid Protein Assay, Quantitative RT-PCR, Negative Control

Journal: bioRxiv

Article Title: Receptor tyrosine kinase AXL regulates Golgi organization and function through an adhesion-Arf1 signaling axis in breast and lung cancers

doi: 10.1101/2025.02.21.639453

Figure Lengend Snippet: (A, B) Representative cross-sectional images of the predominant Golgi phenotype in MDAMB231 cells expressing (A) ManII-GFP (green) or (B) GalTase-RFP (red), treated with DMSO (CNT), or R428 (1μM or 2μM) are shown. Percentage distribution profile for cells (n ≥150) shows organized (white) and disorganized (grey) Golgi in adherent cells across treatments. The graphs represent mean±SEM of percentage distribution profile from three independent experiments. The black bar below the graph represents the gradient of increasing concentration of R428 treatment. (C, D) Representative western blots for (C) S473 phosphorylated Akt (pAkt) and total Akt (Akt) and (D) Y702 phosphorylated AXL (pAXL) and total AXL (AXL) in cell lysates from DMSO (CNT), 0.5μM, 1μM and 2μM R428 treated MDAMB231 cells. Graph represents ratio of densitometric band intensities as mean±SEM from four independent experiments. The black bar below the graph represents the gradient of increasing concentration of R428 treatment. (E) Percentage distribution profile for MCF7 cells (n ≥150) expressing cis-medial Golgi marker ManII-GFP with organized (white) and disorganized (grey) Golgi, in DMSO (CNT), 1μM and 2μM R428 treated cells. Representative cross-sectional confocal images of the predominant Golgi phenotype shown. The graph represents mean±SEM of percentage distribution from three independent experiments. The black bar below the graph represents the gradient of increasing concentration of R428 treatment. (F) Representative western blots for S473 phosphorylated Akt (pAkt) and total Akt (Akt) in cell lysates from DMSO (CNT), 0.5μM, 1μM and 2μM R428 treated MCF7 cells. Graph represents ratio of densitometric band intensities as mean±SEM from three independent experiments. The black bar below the graph represents the gradient of increasing concentration of R428 treatment. (G) Representative western blots for AXL in cell lysates from control (CNT), siAXL1 and siAXL2 treated MDAMB231 cells. Graph represents ratio of densitometric band intensities as mean±SEM from three independent experiments. Representative cross-sectional images of the predominant Golgi phenotype in GM130 (green) immunostained CNT, siAXL1 and siAXL2 MDAMB231 cells. Percentage distribution profile of cells (n ≥150) showing organized (white) and disorganized (grey) Golgi in adherent control (CNT), siAXL1 and siAXL2 treated MDAMB231 cells. The graphs represent mean±SEM of percentage distribution from three independent experiments. Representative western blots for acetylated tubulin and β-tubulin in cell lysates from (H) DMSO (CNT), 0.5μM, 1μM R428 treated and (I) CNT, siAXL1 and siAXL2 treated MDAMB231 cells. Graphs represent ratio of densitometric band intensities as mean±SEM from four and three independent experiments normalized to control respectively. The black bar below the graph represents the gradient of increasing concentration of R428 treatment. Statistical analysis was done using one way ANOVA multiple comparisons test with Tukey’s method for error correction, for the distribution profiles, Mann-Whitney U test for non-normalised and single sample t-test for normalised (with respect to control) western blotting results respectively. All scale bars shown are 10 µm. (*p≤0.05, **p ≤ 0.01, ***p≤ 0.001, ****p ≤ 0.0001, ns= not significant).

Article Snippet: AXL Clone C89E7 (1:2000 for western blotting and 1:400 for immunofluorescence assay) (Cat. No. #8661S) and pAXL D12B2 (Y702) (1:500) (Cat. No. #5724S) was purchased from Cell Signaling.

Techniques: Expressing, Concentration Assay, Western Blot, Marker, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Receptor tyrosine kinase AXL regulates Golgi organization and function through an adhesion-Arf1 signaling axis in breast and lung cancers

doi: 10.1101/2025.02.21.639453

Figure Lengend Snippet: (A) Top Panel - Representative deconvoluted Z-stack maximum intensity projection (MIP) images for AXL (green) and GM130 (red) immunostained stable adherent DMSO (SA-CNT) or R428 (SA-R428) treated MDAMB231 cells. Individual channel and merged images shown. The graph shows the mean±SEM of Pearson’s correlation coefficient for colocalization of AXL and GM130 (n=30 cells) from three independent experiments. (A) Lower Panel – Representative cross-section merged image along with corresponding zoomed image (marked box). Line plot analysis shows fluorescence intensity profile for AXL (green) and GM130 (red) along a solid line in merged MDAMB231 cell image treated with DMSO (SA-CNT) and R428 (SA-R428). (B) Representative western blots for Arf1 and β-tubulin in cell lysates from DMSO (CNT), 0.5μM, 1μM and 2μM R428 treated MDAMB231 cells. Graph represents ratio of densitometric band intensities as mean±SEM from three independent experiments. The black bar below the graph represents the gradient of increasing concentration of R428 treatment. Representative western blots for (C) Arf1 (WB: Arf1) (D) AXL (WB: AXL) in pull down using GST-GGA3 (active Arf1) and in whole-cell lysate (WCL) of DMSO (CNT) and R428 treated MDAMB231 cells. Graph represents ratio of densitometric band intensities in GGA3-PD to WCL as mean±SEM from (C) five (normalized to control) and (D) three independent experiments. (E) Representative western blots for Arf1 (WB: Arf1) in pull down using GST-GGA3 (active Arf1) and in whole-cell lysate (WCL) of control (CNT) and AXL knockdown (using siAXL1 and siAXL2) MDAMB231 cells. Graph represents ratio of densitometric band intensities in GGA3-PD to WCL as mean±SEM from three independent experiments, normalized to control. (F) Representative cross-sectional images and line plot analysis for stable adherent (SA) MDAMB231 cells expressing ABD-GFP (green) and immunostained for AXL (red), treated with DMSO (SA-CNT) or R428 (SA-R428). Individual channel and merged images shown. Line plot profile for lines marked are shown next to their respective images. (G) Representative cross-sectional images of stable adherent MDAMB231 cells expressing constitutively active Arf1 (Q71L-Arf1-mCherry) (red) or wild type-Arf1 (WT-Arf1-mCherry) (red) and cis-medial Golgi marker - ManII-GFP (green) shows the predominant Golgi organization phenotype in DMSO (CNT) and R428 treated cells. Graph shows percentage distribution profile for control and R428 treated WT-Arf1 and Q71L-Arf1 expressing cells (n ≥100) with organized (white) or disorganized (grey) Golgi. The graph represents mean±SEM of percentage distribution from four independent experiments. (H) Representative western blots for AXL and GAPDH in cell lysates from DMSO (CNT), 0.5μM and 1μM Golgicide A (GCA) treated MDAMB231 cells. Graph represents ratio of densitometric band intensities as mean±SEM from three independent experiments. (I) Representative western blots for Y702 phosphorylated AXL (pAXL) and total AXL in cell lysates from DMSO (CNT), 0.5μM Golgicide A (GCA) treated MDAMB231 cells. Graph represents ratio of densitometric band intensities as mean±SEM from four independent experiments. (J) Representative cross-sectional images and line plot analysis for colocalization of AXL (red) and GM130 (green) immunostained adherent MDAMB231 cells, treated with DMSO (CNT) and 1μM GCA for 30 mins. Individual channel and zoomed merged images shown. Line plot profile for lines marked are shown next to their respective images. Statistical analysis was done using one way ANOVA for Pearson’s colocalization analysis and distribution profile. Mann-Whitney U test was used for non-normalised and single sample t test for normalised (with respect to control) western blotting results respectively. All scale bars shown are 10 µm. (*p≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns= not significant).

Article Snippet: AXL Clone C89E7 (1:2000 for western blotting and 1:400 for immunofluorescence assay) (Cat. No. #8661S) and pAXL D12B2 (Y702) (1:500) (Cat. No. #5724S) was purchased from Cell Signaling.

Techniques: Fluorescence, Western Blot, Concentration Assay, Control, Knockdown, Expressing, Marker, MANN-WHITNEY

Journal: bioRxiv

Article Title: Receptor tyrosine kinase AXL regulates Golgi organization and function through an adhesion-Arf1 signaling axis in breast and lung cancers

doi: 10.1101/2025.02.21.639453

Figure Lengend Snippet: (A, B) Representative western blots for (A) Arf1 (WB: Arf1) (B) AXL (WB: AXL) in pull down using GST-GGA3 (GGA3-PD) and in whole-cell lysate (WCL) of stable adherent (SA) and non-adherent (SUS) MDAMB231 cells. Graph represents ratio of densitometric band intensities in GGA3-PD to WCL as mean±SEM from (A) four (normalized to control) and (B) three independent experiments. Representative western blots for (C) Y702 phosphorylated AXL (pAXL-Y702) and AXL (D) S473 phosphorylated Akt (pAkt) and Akt in cell lysates of stable adherent (SA) and non-adherent (SUS) MDAMB231 cells. Graph represents ratio of densitometric band intensities as mean±SEM from (C, D) four independent experiments. (E) Representative deconvoluted maximum intensity projection (MIP) images show immunostained AXL (red) and cis-Golgi GM130 (green), in stable adherent (SA) and non-adherent (SUS) MDAMB231 cells. The graph shows the mean±SEM of Pearson’s correlation coefficient for colocalization of AXL and GM130 (n=30 cells) from three independent experiments. (F) Representative cross-sectional images and line plot analysis for stable adherent (SA) and non-adherent (SUS) MDAMB231 cells expressing ABD-GFP (green) and immunostained for AXL (red). Individual channel and merged images shown. Line plot profile for lines marked are shown next to their respective images. (G) Representative images shown for stable adherent (SA) and non-adherent (SUS) MDAMB231 cells expressing constitutively active Arf1 (Q71L-Arf1-mCherry) (red) or wild type-Arf1 (WT-Arf1-mCherry) (red) and cis-medial Golgi marker ManII-GFP (green). Graph represents the percentage distribution profile of WT-Arf1 and Q71L-Arf1 expressing cells (n ≥150 cells) with organized (white) or disorganized (grey) Golgi. The graph represents mean±SEM from three independent experiments. (H) Representative western blots for acetylated tubulin and β-tubulin in cell lysates of stable adherent (SA) and non-adherent (SUS) MDAMB231 cells. Graph represents ratio of densitometric band intensities as mean±SEM from three independent experiments, normalized to SA. (I) Schematic describes the AXL-Arf1 crosstalk and its regulation of Golgi organization and function downstream of adhesion, adhesion upon R428 treatment (Adhesion+R428) and loss-of adhesion in MDAMB231 cells. It highlights the comparable outcomes R428 treatment has to loss of adhesion. Statistical analysis was done using one-way ANOVA multiple comparisons test with Tukey’s method for error correction, for Pearson’s colocalization analysis and distribution profile. Mann-Whitney U test was used for non-normalised western blotting results and single sample t test for normalised (with respect to control) western blotting results. All scale bars shown are 10 µm. (*p≤0.05, **p ≤ 0.01, ***p≤ 0.001, ****p≤ 0.0001, ns= not significant).

Article Snippet: AXL Clone C89E7 (1:2000 for western blotting and 1:400 for immunofluorescence assay) (Cat. No. #8661S) and pAXL D12B2 (Y702) (1:500) (Cat. No. #5724S) was purchased from Cell Signaling.

Techniques: Western Blot, Control, Expressing, Marker, MANN-WHITNEY

Journal: bioRxiv

Article Title: Receptor tyrosine kinase AXL regulates Golgi organization and function through an adhesion-Arf1 signaling axis in breast and lung cancers

doi: 10.1101/2025.02.21.639453

Figure Lengend Snippet: Representative cross-section images of the predominant Golgi phenotype in (A) A549 and (B) CaLu1 cells expressing cis-medial Golgi marker-ManII-GFP (green) treated with DMSO (CNT) or R428 (1μM or 2μM) are shown. Percentage distribution profile for cells (n ≥100) shows organized (white) and disorganized (grey) Golgi in adherent cells across treatments. The graphs represent mean±SEM from three independent experiments. The black bar below the graph represents the gradient of increasing concentration of R428 treatment. (C, D) Representative western blots for S473 phosphorylated Akt (pAkt) and total Akt (Akt) in cell lysates from DMSO (CNT), 0.5μM, 1μM and 2μM R428 treated (C) A549 and (D) CaLu1 cells. (E, F) Representative western blots for Y702 phosphorylated AXL (pAXL) and total AXL (AXL) in cell lysates from DMSO (CNT), 0.5μM and 1μM R428 treated A549 (E) and CaLu1 (F) cells. Graphs represent ratio of densitometric band intensities as mean±SEM from three or five independent experiments as indicated in graphs. The black bar below the graph represents the gradient of increasing concentration of R428 treatment. (G) Percentage distribution profile for A549 cells (n ≥150) immunostained for cis-Golgi marker -GM130 (green), expressing cis-medial Golgi marker -ManII-GFP (green) or expressing trans-Golgi marker -GalTase-RFP (red) with organized (white) and disorganized (grey) Golgi, in DMSO (CNT) or R428 treated stable adherent (SA) or non-adherent (SUS) cells. Representative deconvoluted z-stack maximum intensity projection (MIP) images of the predominant Golgi phenotype are shown. The graph represents mean±SEM of percentage distribution from three independent experiments. (H) Representative deconvoluted maximum intensity projection (MIP) images show the predominant Golgi organization phenotype, immunostained for cis-Golgi marker GM130 (green) in in control (CNT) and AXL knockdown (siAXL2 treated) stable adherent (SA) vs non-adherent (SUS). Representative western blots for AXL and GAPDH to validate AXL knockdown in cell lysates from CNT vs siAXL2 treated A549 cells. Graph represents mean±SEM of percentage distribution profile for cells (n ≥200 cells) with organized (white) and disorganized (grey) Golgi from three independent experiments. (I) Representative western blot shows the detection of S473 phosphorylated Akt (pAkt) and total Akt (Akt) and total AXL (AXL) and GAPDH in cell lysates from DMSO (CNT) and R428 treated control (CNT) and AXL knockdown (siAXL2) A549 cells. The graphs represent ratio of densitometric band intensities as mean±SEM from three independent experiments. (J) Representative images shown are deconvoluted z-stack maximum intensity projection (MIP), of A549 cells immunostained for AXL (red) and GM130 (green), in non-adherent (SUS) cells treated with DMSO (CNT) or R428. The graph shows the mean±SEM of Pearson’s correlation coefficients for colocalization of AXL and GM130 (n > 20 cells) from three independent experiments. Shown below are cross-section images along with corresponding zoomed image of boxed region marked by dotted line. Line plot shows fluorescence intensity profile along the line for AXL (red) and GM130 stained cis-Golgi (green) in DMSO (CNT) or R428. Statistical analysis was done using one-way ANOVA multiple comparisons test with Tukey’s method for error correction, for the distribution profiles, single sample t test for normalised (with respect to control) western blotting results and Mann-Whitney U test for Pearson’s colocalization analysis. All scale bars shown are 10 µm. (*p≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns= not significant).

Article Snippet: AXL Clone C89E7 (1:2000 for western blotting and 1:400 for immunofluorescence assay) (Cat. No. #8661S) and pAXL D12B2 (Y702) (1:500) (Cat. No. #5724S) was purchased from Cell Signaling.

Techniques: Expressing, Marker, Concentration Assay, Western Blot, Control, Knockdown, Fluorescence, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Receptor tyrosine kinase AXL regulates Golgi organization and function through an adhesion-Arf1 signaling axis in breast and lung cancers

doi: 10.1101/2025.02.21.639453

Figure Lengend Snippet: Representative western blots for (A, B) Arf1 (WB: Arf1) and (C) AXL (WB: AXL) in pull down using GST-GGA3 (GGA3-PD) and whole-cell lysate (WCL) of (A) stable adherent (SA) and non-adherent (SUS) and (B, C) non-adherent (SUS) - DMSO (CNT) and R428 treated A549 cells. Graph represents ratio of densitometric band intensities in GGA3-PD to WCL as mean±SEM from (A, B) four and (C) five independent experiments. Representative western blots for (D, E) Y702 phosphorylated AXL (pAXL), AXL and (F) S473 phosphorylated Akt (pAkt) and Akt in cell lysates of (D) stable adherent (SA) vs non-adherent (SUS) and (E, F) DMSO (CNT) vs R428 treated non-adherent A549 cells. Graph represents ratio of densitometric band intensities as mean±SEM from five or three independent experiments as mentioned in the graphs. (G) Percentage distribution profile for cells (n ≥200 cells) expressing ABD-RFP (red) to detect active Arf1 enriched at an intracellular location, confirmed previously to overlap (white) or not overlap with the Golgi marker (grey) in adherent (SA) and non-adherent (SUS) A549 cells treated with DMSO (CNT) or R428. Representative images are cross-section confocal images showing the predominant Golgi phenotype. The graph represents mean±SEM from three independent experiments. (H) Representative deconvoluted z-stack maximum intensity projection (MIP) images for A549 cells expressing constitutively active Arf1 (Q71L-Arf1-mCherry) (red) or wild-type-Arf1 (WT-Arf1-mCherry) (red), and immunostained for cis-Golgi marker GM130 (green) in DMSO (CNT) and R428 treated non-adherent A549 cells. Graph shows percentage distribution profile for non-adherent control and R428 treated WT-Arf1 and Q71L-Arf1 expressing cells (n ≥200) with organized (white) or disorganized (grey) Golgi. The graph represents mean±SEM from three independent experiments. (I) Representative cross-sectional confocal images with line plot profile for colocalization of immunostained AXL (red) and GM130 (green) or AXL (red) and ManII-GFP (green) expressed in non-adherent A549 cells treated with DMSO (CNT) and 1μM GCA. (J) Representative western blot for acetylated tubulin and total tubulin in cell lysates from DMSO (CNT) and R428 treated - stable adherent (SA) and non-adherent (SUS) A549 cells. The graph represents ratio of densitometric band intensities as mean±SEM from four independent experiments. (K) Graphs represent mean±SEM of median fluorescent intensities of cell surface bound ConA, PNA and WGA lectins, detected by flow cytometry analysis, at early (5 min) or late (120 min) suspension timepoints and in DMSO (CNT) and R428 treated A549 cells from three independent experiments. (L) Representative images of crystal violet stained colonies in soft agar of DMSO (CNT) and R428 treated A549 cells. The graph represents mean±SEM of number of colonies counted from 20 images obtained from two independent experiments. (M) Schematic describes the AXL-Arf1 crosstalk and its regulation of Golgi organization and function downstream of adhesion, loss of adhesion and loss of adhesion on R428 treatment (loss of adhesion+R428) in A549 cells. Statistical analysis was done using one-way ANOVA multiple comparisons test using Tukey’s method for error correction for the distribution profiles, Mann-Whitney U test for non-normalised western blotting results and AIG assay, and single sample t-test for normalised (with respect to early suspension time point and control) flow cytometry results. All scale bars shown are 10 µm. (*p≤0.05, **p≤ 0.01, ***p≤ 0.001, ****p≤ 0.0001, ns= not significant).

Article Snippet: AXL Clone C89E7 (1:2000 for western blotting and 1:400 for immunofluorescence assay) (Cat. No. #8661S) and pAXL D12B2 (Y702) (1:500) (Cat. No. #5724S) was purchased from Cell Signaling.

Techniques: Western Blot, Expressing, Marker, Control, Flow Cytometry, Suspension, Staining, MANN-WHITNEY