Human Phospho-Axl (Y779) Antibody

Detection of Human Axl by Western Blot

Migration of H1299 NSCLC cells enhanced by ligand-dependent Axl activation. (A) Western blotting to assess Gas6 expression in H1299 cells. Expression of Gas6 in LCAFhTERT cells was used as a positive control. (B) Phosphorylation of Axl was analyzed by Western blotting of whole cell lysates using different antibodies. GAPDH was used as an internal control. H1299 cells were stimulated for 15 min with 400 nM recombinant human Gas6 (rGas6). H1299 cells were treated with or without TP-0903 (0.2 µmol/L) for 24 h. (C) Migration of H1299 cells was analyzed using rGas6 (400 nM) added to the lower chamber. H1299 cells were treated with or without TP-0903 (0.2 µmol/L) for 24 h. (D) Western blotting of conditioned medium from LCAFhTERT transfected with siGas6 or siScr (control) to assess whether they contains Gas6 secreted by CAFs. (E) Phosphorylation of Axl in H1299 cells analyzed by Western blotting after stimulation with conditioned medium from siRNA-transfected LCAFhTERT. H1299 cells were treated with or without TP-0903 (0.2 µmol/L) for 24 h. The medium (DMEM containing 10% FBS) was used as control. (F) Migration of H1299 cells analyzed using conditioned medium of siRNA-transfected LCAFhTERT. H1299 cells were treated with or without TP-0903 (0.2 µmol/L) for 24 h. The medium (DMEM containing 10% FBS) was used as control. The relative number of migrated H1299 cells is indicated on the y-axis. Data show the mean ± SEM (n = 3); **p < 0.01. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28878389), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Axl by Western Blot

Migration of H1299 NSCLC cells enhanced by ligand-dependent Axl activation. (A) Western blotting to assess Gas6 expression in H1299 cells. Expression of Gas6 in LCAFhTERT cells was used as a positive control. (B) Phosphorylation of Axl was analyzed by Western blotting of whole cell lysates using different antibodies. GAPDH was used as an internal control. H1299 cells were stimulated for 15 min with 400 nM recombinant human Gas6 (rGas6). H1299 cells were treated with or without TP-0903 (0.2 µmol/L) for 24 h. (C) Migration of H1299 cells was analyzed using rGas6 (400 nM) added to the lower chamber. H1299 cells were treated with or without TP-0903 (0.2 µmol/L) for 24 h. (D) Western blotting of conditioned medium from LCAFhTERT transfected with siGas6 or siScr (control) to assess whether they contains Gas6 secreted by CAFs. (E) Phosphorylation of Axl in H1299 cells analyzed by Western blotting after stimulation with conditioned medium from siRNA-transfected LCAFhTERT. H1299 cells were treated with or without TP-0903 (0.2 µmol/L) for 24 h. The medium (DMEM containing 10% FBS) was used as control. (F) Migration of H1299 cells analyzed using conditioned medium of siRNA-transfected LCAFhTERT. H1299 cells were treated with or without TP-0903 (0.2 µmol/L) for 24 h. The medium (DMEM containing 10% FBS) was used as control. The relative number of migrated H1299 cells is indicated on the y-axis. Data show the mean ± SEM (n = 3); **p < 0.01. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28878389), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Axl by Western Blot

DHA treatment inhibits Axl expression and proteins involved in the Axl signaling pathway, reduces cell migration and invasion and induces apoptosis in mCRPCa cell lines.a qRT-PCR for Axl in PCa cells treated with 5 μM of DHA for 24 h. Values were normalized to Gapdh levels and to DMSO control. The experiment was performed in triplicate. Data are representative of 3 independent experiments; *p < 0.05 two-tailed Student’s t-test. b Immunoblot analysis of protein extracts obtained from DU145 and PC-3 treated with 5 μM DHA for 24 h using anti-phospho-Axl, anti-phospho-Akt and anti-phospho-Stat3 and anti-Axl, anti-Akt, anti-Stat3 and anti- GAPDH. c Migration and d invasion analysis of mCRPC cell lines 24 h post treatment with 5 μM of DHA. Cells were fixed and stained, and 3–5 random microscopic fields were counted. Data are representative of three independent experiments and all values shown are mean ± SD from a representative experiment; **p < 0.01, two-tailed Student’s t-test. e Apoptosis assay for mCRPCa cell lines treated with 5 μM of DHA for 8 h. DMSO 0.05% was used as a control. Data are representative of three independent experiments and all values shown are mean ± SD from a representative experiment; *p < 0.05, two-tailed Student’s t-test Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30783079), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-Axl (Y779) by Western Blot

The effects of ProS1, Gas6 and chimeras on TAM receptor and coupled downstream signalling molecule activation in SCC-25 cells. (a) Schematic representation of recombinant TAM ligand constructs used in this study. These included human ProS1, Gas6, and three ProS1/Gas6 chimeras. All of the chimeras contained the Gla domain and EGF-like domains of ProS1. Light grey colour denotes regions corresponding to ProS1 amino acid sequence, whereas dark grey denotes regions corresponding to Gas6 amino acid sequence. (b) Western blot showing phosphorylated Tyro3 (pTyro3) and Erk (pErk) levels after stimulation with recombinant Gas6, ProS1 and three chimeras (7.5 nM) for 9 min. (c) Western blot showing phosphorylated Axl (pAxl) and Akt (pAkt) levels under the same experimental conditions as in (b). Each representative blot image is followed by accompanying graphs of densitometric quantification of bands (n = 3 separate experiments). Data as mean ± SEM expression for each phosphoprotein was normalized against the total protein/loading control (tTyro3, tERK, GAPDH, actin). ANOVA with Tukey's multiple comparison post-hoc analysis; ***p < 0.001, **p < 0.01, *p < 0.05 versus control (untreated). While the sample loading order is different in the pAxl blot, the quantification bar charts are presented in the same order for consistency. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35518197), licensed under a CC-BY license. Not internally tested by R&D Systems.