primary antibodies against human tyro3  (Cell Signaling Technology Inc)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Viral Infection sensitizes Human Fetal Membranes to Bacterial LPS by MERTK Inhibition and Inflammasome Activation 1

doi: 10.4049/jimmunol.1700870

Figure Lengend Snippet: Primers used for qRT-PCR

Article Snippet: TYRO3, AXL, and MERTK levels were evaluated by Western blot as previously described ( 8 ) using the anti-human primary antibodies against TYRO3 (#MAB859, R&D Systems), AXL (#AF154, R&D Systems), and MERTK (#AF891, R&D Systems).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Viral Infection sensitizes Human Fetal Membranes to Bacterial LPS by MERTK Inhibition and Inflammasome Activation 1

doi: 10.4049/jimmunol.1700870

Figure Lengend Snippet: (A) Untreated FM explants (n=3) were homogenized and analyzed for expression of TYRO3, AXL, MERTK, GAS6 and PROS1 mRNA by qRT-PCR. (B–C) Human FM explants were treated with no treatment (NT), LPS (100ng/ml), MHV-68 (1.5×104/ml PFU) or both MHV-68 and LPS in the presence of media or rGas6 (50ng/ml) (n=5). Tissues were homogenized for protein and Western blot performed for (B) AXL (~140kDa) and (C) MERTK (~180kDa). Blots are from one representative experiment. Bar charts show AXL and MERTK expression as determined by densitometry and normalized to b-actin. (D) FM explants were treated with NT, LPS (1ng/ml), Poly(I:C) (20µg/ml) or both Poly(I:C) and LPS (n=4). Tissues were homogenized for protein and Western blot performed for AXL and MERTK. Blots are from one representative experiment. Bar charts show AXL and MERTK expression as determined by densitometry and normalized to β-actin. (E–F) Human FM explants were treated with no treatment (NT), LPS, MHV-68 or both MHV-68 and LPS in either the presence of media or rGAS6. Tissues were homogenized for protein and ELISA performed for (E) sMERTK (n=7); (F) GAS6 (n=5), and (G) PROS1 (n=8). *p<0.05 relative to the NT control unless otherwise indicated. Data are expressed as mean±SEM.

Article Snippet: TYRO3, AXL, and MERTK levels were evaluated by Western blot as previously described ( 8 ) using the anti-human primary antibodies against TYRO3 (#MAB859, R&D Systems), AXL (#AF154, R&D Systems), and MERTK (#AF891, R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Viral Infection sensitizes Human Fetal Membranes to Bacterial LPS by MERTK Inhibition and Inflammasome Activation 1

doi: 10.4049/jimmunol.1700870

Figure Lengend Snippet: (A) Human FM explants were treated with no treatment (NT), LPS (100ng/ml), MHV-68 (1.5×104/ml PFU) or both MHV-68 and LPS in the presence of media or rGas6 (50ng/ml). (i) IL-1β secretion in supernatants was measured by ELISA (n=6). Lysates were evaluated by Western blot for expression of (ii) pro-IL-1β and (iii) active IL-1β. Bar charts show pro- and active-IL-1β expression as determined by densitometry and normalized to β-actin (n=4–5) *p<0.05 relative to the NT control unless otherwise indicated. (B) Human FM explants were treated with or without LPS (1ng/ml) in the presence of blocking anti-TAM Abs or isotype control Abs. IL-1β secretion was measured and levels expressed as LPS-induced fold change (FC) relative to the NT control (n=4; *p<0.05 relative to the isotype control). (C–D) Pregnant wildtype or AXL−/−MERTK−/− mice were injected on E15.5 with either PBS or LPS (20µg/kg). After 6hrs mice were sacrificed. The FMs were harvested and homogenized for RNA. (D) TYRO3, AXL, MERTK, GAS6 and PROS1 mRNA expression levels in FMs from PBS treated wildtype mice (n=3) were measured by qRT-PCR. (E) IL-1B mRNA expression levels in FMs from PBS and LPS treated wildtype or AXL−/−MERTK−/− mice were measured by qRT-PCR. *p<0.05 relative to the control groups unless otherwise indicated. Data are expressed as mean±SEM.

Article Snippet: TYRO3, AXL, and MERTK levels were evaluated by Western blot as previously described ( 8 ) using the anti-human primary antibodies against TYRO3 (#MAB859, R&D Systems), AXL (#AF154, R&D Systems), and MERTK (#AF891, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control, Blocking Assay, Injection, Quantitative RT-PCR

Journal: British Journal of Cancer

Article Title: Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma

doi: 10.1038/bjc.2017.354

Figure Lengend Snippet: Characterisation of TAM receptor expression in a panel of human LMS cell lines. ( A ) Lysates from IB112, IB118, IB133, IB134, IB136, SK-LMS-1 and HepG2 (positive control) were analysed by western blot. Beta-actin was used as loading control. Longer exposition is shown for HepG2 with AXL antibody. ( B ) Leiomyosarcoma cells were starved then stimulated with GAS6. After normalisation for protein concentration, phosphotyrosine proteins were immunoprecipitated with a PY20 antibody. The SK-LMS-1 lysate was immunoprecipitated also with an antibody isotype control. Western blot analysis was performed with an anti TYRO3 antibody. ( C ) Graph showing AXL (red), MER (blue) and TYRO3 (green) expression levels in LMS and HepG2 cells by FACS analysis. Results are shown as fold increase compared to isotype control. ( D ) Evaluation of GAS6 levels in LMS cells lysates by ELISA.

Article Snippet: Primary antibodies against human TYRO3 (#5585; Cell Signalling, Saint Quentin Yvelines, France), AXL (ABN275; Millipore, Molsheim, France), phosphoAXL (Y779-AF2228; R&D Systems) and actin (A54441; Sigma-Aldrich) were used, followed by a secondary HRP-linked antibody.

Techniques: Expressing, Positive Control, Western Blot, Control, Protein Concentration, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: British Journal of Cancer

Article Title: Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma

doi: 10.1038/bjc.2017.354

Figure Lengend Snippet: (A) TYRO3 and AXL RTK protein expression following shRNA knockdown. SK-LMS-1 cells comprising TYRO3-targeting shRNA (sh1 and sh2), AXL-targeting shRNA (sh3 and sh4) and control shRNA (shPRPC) show TYRO3 and AXL levels by immunoblotting. (B) Knockdown of TYRO3 and AXL in the SK-LMS-1 cell line affects cell viability. Graph showing the reduction of viability in SK-LMS-1 knocked-down cells cultivated for 3 days. Viable cells were counted using trypan blue. Graphs represent means of two independent experiments performed in duplicates. (C) Graphs showing decrease in colony formation potential for SK-LMS-1 knockdown cells. Graphs represent means of two independent experiments performed in duplicates.

Article Snippet: Primary antibodies against human TYRO3 (#5585; Cell Signalling, Saint Quentin Yvelines, France), AXL (ABN275; Millipore, Molsheim, France), phosphoAXL (Y779-AF2228; R&D Systems) and actin (A54441; Sigma-Aldrich) were used, followed by a secondary HRP-linked antibody.

Techniques: Expressing, shRNA, Knockdown, Control, Western Blot

Journal: British Journal of Cancer

Article Title: Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma

doi: 10.1038/bjc.2017.354

Figure Lengend Snippet: Crizotinib and foretinib deactivate TYRO3 and AXL phosphorylation and lead to decrease in cell viability. ( A ) Proteins with phosphorylated tyrosines were immunoprecipitated with a PY20 antibody from LMS cell lysates treated with crizotinib and foretinib. IB118 and SK-LMS-1 were precipitated also with an isotype control antibody, as shown in the right part of the panel. Western blot analysis was performed with an anti-TYRO3 and -AXL antibodies. Graphs showing TYRO3 ( B ) or AXL ( C ) protein quantification of western blot using ChemiDoc Imaging Systems (Bio-Rad). Leiomyosarcoma cells were treated with crizotinib ( D ) and foretinib ( E ) at indicated concentrations for 72 h. Viable cells were measured using CellTiterGlo (Promega) and plotted relative to untreated control. Graphs represent means of three independent experiments performed in triplicates. Bars represent s.d.’s.

Article Snippet: Primary antibodies against human TYRO3 (#5585; Cell Signalling, Saint Quentin Yvelines, France), AXL (ABN275; Millipore, Molsheim, France), phosphoAXL (Y779-AF2228; R&D Systems) and actin (A54441; Sigma-Aldrich) were used, followed by a secondary HRP-linked antibody.

Techniques: Phospho-proteomics, Immunoprecipitation, Control, Western Blot, Imaging

Journal: British Journal of Cancer

Article Title: Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma

doi: 10.1038/bjc.2017.354

Figure Lengend Snippet: TYRO3, AXL and GAS6 expression in sarcomas. ( A ) Representative example of immunohistochemistry on LMS tissues with anti-AXL, anti-TYRO3 and anti-GAS6 antibodies. TYRO3 nuclear expression is shown in detail. Original magnification × 20 and × 40. ( B ) Graph showing the percentage of positive samples from each sarcoma hystotype analysed. Cytoplasmic or nuclear staining was plotted separately. ( C – F ) Gene expression analysis of TYRO3, AXL, GAS6 and PROS1 transcripts, respectively. Data are from gene expression and outcome in 251 sarcoma patients’ samples from ATGsarc microarray database. ( G ) Kaplan–Meier curve for progression-free survival of 94 LMS patients with low or mixed and high expression levels of GAS6 and PROS1 genes cluster. P -values in log-rank test are indicated. n =number of patients in each group. Median PFS are expressed in years.

Article Snippet: Primary antibodies against human TYRO3 (#5585; Cell Signalling, Saint Quentin Yvelines, France), AXL (ABN275; Millipore, Molsheim, France), phosphoAXL (Y779-AF2228; R&D Systems) and actin (A54441; Sigma-Aldrich) were used, followed by a secondary HRP-linked antibody.

Techniques: Expressing, Immunohistochemistry, Staining, Gene Expression, Microarray