Mammary tumors were minced and resuspended in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Deoxycholate, 0.1% SDS and 50 mM Tris HCl, pH 8) with protease inhibitors, sonicated, spun down, and fat was removed before protein quantification. Immunoblots were performed using 20–60 μg of protein using the following antibodies, all from Cell Signaling Technology (Danvers, MA): HER2 (#4290T), p-HER2 (Y1248, #2247T), p-HER2 (Y1221/1222, #2243T), Akt (#4691T), p-Akt (S473, #4060T), p-Akt (T308, #13038T), actin (#3700T). The mouse phospho-RTK analysis was performed using the Proteome Profiler Mouse Phospho-RTK Array Kit (R&D Systems, #ARY014, Minneapolis, MN) according to the manufacturer’s instructions. Signal densities were quantified using NIH Image software and normalized as indicated in the figure legends and also, to the background.
Proteome profiler mouse phospho rtk array kit
Manufactured by R&D Systems
Sourced in United States, Germany
The Proteome Profiler Mouse Phospho-RTK Array Kit is a multiplex array that allows for the simultaneous detection and comparison of the phosphorylation status of multiple receptor tyrosine kinases (RTKs) in mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Tubulin sc 5286, by Santa Cruz Biotechnology (2 mentions)
Lamin a c 4777, by Cell Signaling Technology (2 mentions)
Phospho ron y1238 y1239 af1947, by R&D Systems (2 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
Pierce ecl 2 western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Proteome profiler mouse xl cytokine array, by R&D Systems (2 mentions)
P her2, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ultrasonification, by Sonics (1 mentions)
Cell recovery solution, by Corning (1 mentions)
Chemiluminescence detection system, by Cytiva (1 mentions)
Molecular imager gs 800, by Bio-Rad (1 mentions)
Quantity one 1 d analysis software v4, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti vinculin, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
19 protocols using proteome profiler mouse phospho rtk array kit
1
Characterization of HER2 and Akt Signaling in Mammary Tumors
2
Quantifying RTK Phosphorylation
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RTK phosphorylation was measured using Proteome Profiler Mouse Phospho-RTK array Kit (R & D Systems, # ARY014) as instructed by the manufacturers.
3
Phospho-RTK Array for Uterine Tissue
4
Protein Extraction and Western Blot Analysis
5
Western Blot Analysis of Signaling Pathways
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Cells were homogenized in RIPA buffer as described.17 Nuclear and cytoplasmic extracts were isolated by centrifugation and hypotonic lysis as described.18 Antibodies for analyses included: RON (SC‐322), Androgen Receptor, (SC‐815), Axl (SC‐1096), Gas6 (SC‐1935), and Tubulin (SC‐5286) from Santa Cruz Biotechnology; Src (2110 S), phosphor‐Src y416 (2101 S), Akt (4691 S), phosphor‐Akt s473 (4060S), phospho‐Axl y702 (5724S), and LAMIN A/C (4777S) from Cell Signaling Technologies; phospho‐RON y1238/y1239 (AF1947, R&D); ACTIN (Cincinnati Children's Hospital Medical Center, Clone C4). Peroxidase‐conjugated secondary antibodies (Jackson Laboratories) were applied, and membranes were developed using Pierce ECL2 Western Blotting substrate (ThermoFisher Scientific). Membranes were stripped using Restore Western Blot Stripping Buffer (ThermoFisher Scientific) before re‐probing. The Proteome Profiler Mouse Phospho‐RTK Array Kit (ARY014, R&D) was used on whole tumor lysates and performed according to the manufacturer's instructions.
6
HGF-Induced RTK Activation Assay
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Cells were seeded at 3 × 105 per well in 12‐well plates and cultured overnight. After serum starvation for 4 hours, cells were stimulated with HGF for 10 minutes, followed by washing with ice‐cold PBS, lysis in 100 μL of 1 × SDS‐PAGE Laemmli sample buffer (FUJIFILM Wako Pure Chemical), and ultrasonification (Sonics & Materials Inc., Newtown, CT, USA). Cell lysates were analyzed by SDS‐PAGE with a 10% polyacrylamide gel and electroblotted onto a PVDF membrane (Bio‐Rad, Hercules, CA, USA). The membrane was treated with primary antibodies (1:1000), followed by HRP‐conjugated secondary antibodies (Dako, Carpinteria, CA, USA) (1:2000). Chemiluminescence was visualized and quantitated using ImmunoStar LD (FUJIFILM Wako Pure Chemical). For RTK array analysis, cell lysates containing 800 μg protein were analyzed using Proteome Profiler Mouse Phospho‐RTK Array Kit (R&D Systems, Minneapolis, MN, USA), in accordance with the manufacturer's instructions.
7
Receptor Tyrosine Kinase Profiling in Mouse Brain
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Mice were transcardially perfused with PBS, brains removed, and a 3-mm-thick tissue slice (+ 2.50 ± 0.5 to 0.00 ± 0.5 mm relative to bregma) was prepared from each brain and separated into the left and right hemispheres. The relative level of tyrosine phosphorylation of 39 different receptor tyrosine kinases (RTK) was determined in brain tissue samples using the Proteome Profiler Mouse Phospho-RTK Array Kit (R&D Systems, Wiesbaden, Germany, #ARY014) according to manufacturer’s instructions. Briefly, 500 μl lysis buffer was added to each brain tissue slice, and tissue samples were homogenized mechanically as described above. Following incubation on ice for 10 min, tissue homogenates were centrifuged for 5 min at 16,100×g at 4 °C. Protein concentration in the supernatant was then quantified by Bradford assay, and 250 μg protein/sample was processed further following the protocol of manufacturer.
8
Profiling Phospho-RTK and Cytokines
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Mouse prostate tumors treated with defined agents were processed as instructed by manufacturer’s protocol and an equal amount (μg) of lysate was used to quantify phospho-RTK proteins with Proteome Profiler™ Mouse Phospho-RTK Array Kit (R&D Systems, ARY014) or Proteome Profiler Mouse XL Cytokine Array (R&D Systems, ARY028). Medium conditioned by CPPSML PCa cell lines was analyzed with Proteome Profiler Mouse Cytokine Array Kit – Panel A (R&D Systems, ARY006). Quantification of the spot intensity in the arrays was conducted with background subtraction in ImageJ.
9
Serum-Starved Astrocyte Phospho-RTK Analysis
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Astrocytes were serum-starved overnight and then analyzed using the Proteome Profiler Mouse Phospho-RTK Array Kit (R&D) following the manufacturer’s instructions.
10
Analyzing Organoid Response to Kinase Inhibitors
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