The expression of TIM-1, Axl, and MXRA8 was analyzed by staining cells with anti-TIM-1, anti-Axl (R&D Systems, Minnneapolis, MN, USA), and anti-MXRA8 (JSR life sciences, Sunnyvale, CA, USA) monoclonal antibodies without prior fixation. The primary staining with unconjugated antibody was followed by secondary staining with either Alexa 488- or 647-conjugated anti-mouse/goat IgG antibody (ThermoFisher, Waltham, MA, USA). The respective IgG isotype was used as control. The expression of TIM-1 and Axl was analyzed by APC-conjugated monoclonal anti-TIM-1 (BioLegend®) and anti-Axl (R&D Systems, Minnneapolis, MN, USA) antibodies respectively. Appropriate APC-conjugated isotype control antibodies from BioLegend® and R&D Biosystems were used. All flow cytometry analyses in this study were performed using Sony Spectral Cell Analyzer (Sony Biotechnology, San Jose, CA, USA) and data analyzed by FlowJo V10 Software.
Anti axl
Manufactured by R&D Systems
Sourced in United States, Japan
Anti-Axl is a recombinant protein that can be used in research applications. It functions as a ligand for the Axl receptor tyrosine kinase.
Automatically generated - may contain errors
Lab products found in correlation
Anti actin, by Merck Group (3 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti mertk, by R&D Systems (2 mentions)
Anti zeb1, by Santa Cruz Biotechnology (2 mentions)
Anti snail, by Cell Signaling Technology (2 mentions)
Anti p axl y702, by Cell Signaling Technology (2 mentions)
Anti slug, by Cell Signaling Technology (2 mentions)
Anti tim 1, by BioLegend (1 mentions)
Anti tim1, by R&D Systems (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions)
Anti src, by Cell Signaling Technology (1 mentions)
Anti phospho src, by Cell Signaling Technology (1 mentions)
Anti phospho axl, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti tyro3, by R&D Systems (1 mentions)
Anti gas6, by R&D Systems (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
17 protocols using anti axl
1
Multiparameter Flow Cytometry Analysis
2
Immunoblotting Quantification and Normalization
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Immunoblotting was performed as described previously [30 (link)]. The primary antibodies were used at a dilution of 1:1000 if not indicated otherwise: anti-phospho-Axl (#5724), anti-phospho-Erk1/2 (#9101), anti-Erk1/2 (#4695), anti-phospho-Src (#6943) and anti-Src (#2109) were purchased from Cell Signaling (Danvers, MA, USA). Anti-Axl (#AF154) and anti-PRAME (#ab219650) were obtained from R&D Systems (Minneapolis, MN, USA) and Abcam (Cambridge, England, UK), respectively. Anti-actin (#A2206, Merck, Darmstadt, Germany) was applied at 1:5000 as a loading control. Horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories, Newark, CA, USA) were diluted 1:10.000. Abundance of proteins was quantified via densitometry using ImageJ software (https://imagej.nih.gov/ij/ ). Peak areas were quantified and normalized to the actin loading control. For the control versus the treatment experiments, the abundance of proteins upon interference was set relative to the control.
3
Quantitative Protein Analysis of Corpus Callosum
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For western blotting, corpus callosum tissue of mice was quickly resected on ice and ultrasonically homogenised in RIPA buffer supplemented with 1 mM PMSF and protease inhibitor cocktail. Equal amounts of protein samples (∼20 μg) from each group were loaded in SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with the following primary antibodies: antimyelin basic protein (MBP) (1 : 400, Merck Millipore), anti-MerTK (1 : 1000, R&D Systems), anti-Axl (1 : 1000, R&D Systems), anti-Tyro3 (1 : 1000, R&D Systems), anti-Gas6 (1 : 1000, R&D Systems), and anti-β-actin (1 : 10000, ProteinTech). Protein expression was detected using an enhanced chemiluminescence detection system (Tanon) via incubation with peroxidase-conjugated secondary antibodies. Images were obtained using an ImageQuant LAS4000 mini image analyser (GE Healthcare).
4
Western Blot Analysis of AXL Protein
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Cells were washed twice in ice-cold PBS and lysed in lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 5 mM EGTA, 1 mM Na3VO4, and 1× protease inhibitor cocktail).
Protein concentration was determined by the Bradford assay (Bio-Rad, Segrate, MI, Italy) using BSA as the standard, considering that 5 μg of BSA shows a value of absorbance of 0.333 at a wavelength of 595 nm. Equal amounts of protein were denatured in sample buffer (10% SDS, 87% glycerol, 5% β-mercaptoethanol and 0.1% bromophenol blue) for 5 min at 100°C, and then analyzed by SDS-PAGE (10% acrylamide).
Gels were electroblotted onto nitrocellulose membranes (G&E Healthcare, Milan, MI, Italy) and, after the transfer, membranes were blocked for 1 hr with a solution of 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20.
Filters were probed with the primary antibodies with shaking overnight at 4°C. Detection was performed by peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system (Thermo Fisher, Life Technologies, Monza, MB, Italy). Primary antibodies used were anti-AXL (R&D Systems, Milan, MI, Italy) and anti-β-actin (Sigma Aldrich, Milan, MI, Italy).
Protein concentration was determined by the Bradford assay (Bio-Rad, Segrate, MI, Italy) using BSA as the standard, considering that 5 μg of BSA shows a value of absorbance of 0.333 at a wavelength of 595 nm. Equal amounts of protein were denatured in sample buffer (10% SDS, 87% glycerol, 5% β-mercaptoethanol and 0.1% bromophenol blue) for 5 min at 100°C, and then analyzed by SDS-PAGE (10% acrylamide).
Gels were electroblotted onto nitrocellulose membranes (G&E Healthcare, Milan, MI, Italy) and, after the transfer, membranes were blocked for 1 hr with a solution of 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20.
Filters were probed with the primary antibodies with shaking overnight at 4°C. Detection was performed by peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system (Thermo Fisher, Life Technologies, Monza, MB, Italy). Primary antibodies used were anti-AXL (R&D Systems, Milan, MI, Italy) and anti-β-actin (Sigma Aldrich, Milan, MI, Italy).
5
Quantifying ACE2, NRP1, and AXL Proteins
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Cell lysates were extracted in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher). The protein concentrations were determined by a bicinchoninic acid (BCA) assay (Thermo Fisher), and the proteins were subjected to 4 to 12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat dried skim milk and incubated with the specific antibodies anti-ACE2 (catalog number AF933; R&D) (1:1,000), anti-NRP1 (catalog number MA5-32179; Thermo Fisher) (1:1,000), and anti-AXL (catalog number AF154; R&D) (1:1,000). Protein bands were detected using an enhanced chemiluminescence image analyzer. β-Actin (catalog number A5316; Millipore-Sigma) (1:5,000) was used as an internal control to assess the comparability of protein loading.
6
Immunohistochemical Analysis of Myelination
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Mice were anaesthetised with an intraperitoneal injection of pentobarbital sodium and perfused with cold saline solution followed by 4% formaldehyde. Their brains were dissected and then immersed in 4% formaldehyde for fixation followed by dehydration with sucrose solution. The coronal brain sections used for histological analysis were derived mostly from the caudal callosum, beginning at approximately −0.5 mm from bregma. The brain sections (25 μm) were first washed three times with PBS and then blocked with 4% goat serum in 0.3% Triton X-100 for 1 h at room temperature. Subsequently, they were incubated overnight at 4°C with the anti-MBP (1 : 400, Merck Millipore), anti-Iba1 (1 : 400, Wako, Japan), anti-MerTK (1 : 100, R&D Systems), or anti-Axl (1 : 100, R&D Systems) primary antibodies. Samples were then stained with Alexa Fluor 488 or 594 secondary antibodies (1 : 1000, Invitrogen) at room temperature for 2 h before rinsing with PBS. Sections were mounted with fluorescence decay resistant medium (with DAPI) before they were covered with a cover slip. Images were captured using a confocal microscope (Olympus FluoView FV1000).
7
Immunoblotting of Axl and Actin
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Immunoblotting was done as described (31 (link)). In brief, blotted membranes were blocked in 4% bovine serum albumin (BSA) in TBS-T (0.1%). The primary anti-Axl (R&D Systems; #AF154) and anti-actin (Sigma; #A2066) were diluted 1:1000 in blocking solution and incubated overnight at 4°C.
8
ZIKV Entry Inhibition via Axl Blockade
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ZIKV containing Nano luciferase gene (ZIKV-Nluc), generated using strategy previously reported (45 (link)), was used to evaluate ZIKV entry into SC after Axl receptor blockade. SC, seeded in 96-well solid white microplates, were preincubated with 10 μg/ml anti-Axl (catalog no. AF154; R&D Systems), 1 μM R428, or respective goat IgG (50 μg/ml) (catalog no. AB-108-C; R&D Systems) or DMSO vehicle (1:10,000 dilution) controls for 2 h at 37°C prior to infection. SC were then infected with ZIKV-Nluc at different MOIs for 1 h in the presence of antibody or drug, washed twice with PBS, and replaced with fresh media and drug. At 4 and 24 h postinfection, the cells were washed twice with PBS, and luciferase activity (luminescence) was determined using Nano-Glo luciferase assay system (catalog no. N1150; Promega). Luciferase activity is reported as percent relative luminescence of matched time point controls.
9
Protein Expression Analysis in Cell Lysates
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Whole-cell lysates were prepared using RIPA buffer as described previously [19 (link)] and analyzed using the following primary antibodies: anti-uPA, anti-c-Src, anti-ZEB1, anti-β-actin, and anti-GAPDH (Santa Cruz Biotechnology); anti-vimentin (Sigma, St Louis, MO, USA); anti-myc (Upstate Biotechnology, Lake Placid, NY, USA); anti-Slug, anti-Snail, anti-cyclin D1, anti-phospho-c-Jun(S63), anti-c-Jun, anti-phospho-ATF-2(T71), anti-phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-ERK1/2, anti-phospho-c-Src(Y416), anti-phospho-JNK(T183/Y185), and anti-JNK (Cell Signaling Tech., Danvers, MA, USA); anti-Twist1 (Abcam, Cambridge, MA, USA); anti-ZEB2 (Active Motif, Tokyo, Japan); anti-Axl (R&D systems, Minneapolis, MN, USA); anti-TMPRSS4 (in-house) [19 (link)]. Where indicated, cells were transiently transfected for 24 h and then treated with 40 mM PD98059, 15 mM SP600125, 3 mM SU6656 (Sigma), or 0.4% dimethyl sulfoxide (DMSO) for 24 h before lysate preparation.
10
miRNA Regulation of Cell Signaling Pathways
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miR precursors: Pre-miR™ miRNA Precursor Negative Control #1, Pre-miR™ miRNA Precursor hsa-miR-148b (PM10264) (Applied Biosystems). TaqMan® MicroRNA assays for miRNA detection: Hsa-miR-148b-3p ID 000471, U6 snRNA ID001973, U44 snRNA ID001904 (Applied Biosystems). Quantitect Primer Assay: 218300Axl ID 33000 (Qiagen). Qiagen miScript-SYBR Green PCR Kit and miScript Primer Assay: hsa-let-7g ID 1 (Qiagen). Primary antibodies: anti-Cleaved Caspase-3 (Asp175) #9661 (Cell Signaling Technology), anti-Ki67 ab15580 (Abcam), anti-ITGA5 pAb RM10 kindly provided by G. Tarone laboratory (Molecular Biotechnology Center, University of Torino), anti-CD166/ALCAM mAb MOG/07 (Novocastra Laboratories), anti-GAPDH pAb V-18, anti-ACTIN I-19 pAb (all form Santa Cruz Biotechnology), anti-AXL (R&D Systems). Secondary antibodies: HRP-conjugated goat anti-mouse IgG, goat anti-rabbit IgG (Santa Cruz Biotechnology).
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