The Human Phospho-MAPK Array Kit (Cat# ARY002B; R&D System, Minneapolis, MN, USA) was used according to the manufacturer’s instructions to identify proteins phosphorylated upon AcGal-1 treatment. Briefly, 2 ml of Array Buffer 5 was pipetted into each well of a 4-well plate, which was then incubated for 1 h on a rocking platform shaker. Samples were prepared by adding up to 400 µl of sample to separate tubes. The final volume was adjusted to 1.5 ml with Array Buffer 1. A 20 µl volume of reconstituted detection antibody cocktail was added to each sample, followed by mixing and incubation at room temperature for 1 h. Array Buffer 5 was removed by aspiration and the prepared sample/antibody mixture was incubated overnight at 2 °C on a rocking platform shaker. After washing the membrane with 1× wash buffer for 10 min on the shaker, 2 ml of diluted streptavidin-horseradish peroxidase was pipetted into each well followed by incubation for 30 min at room temperature. Finally, 1 ml of Chemi Reagent Mix was pipetted onto the membrane, which was exposed to X-ray film for 1–10 min.
Human phospho mapk array kit
Manufactured by R&D Systems
Sourced in United States
The Human Phospho-MAPK Array Kit is a multiplex ELISA-based assay designed to detect and quantify the relative phosphorylation of 26 different mitogen-activated protein kinases (MAPKs) in human samples. The kit provides a platform for simultaneous analysis of these key signaling proteins, enabling researchers to profile the activation status of multiple MAPK pathways in a single experiment.
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Lab products found in correlation
Human phospho rtk array kit, by R&D Systems (4 mentions)
Mouse igg peroxidase conjugate, by Merck Group (1 mentions)
P p38 mapk, by Cell Signaling Technology (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Chemiluminescence gel imaging system, by Bio-Rad (1 mentions)
Nu7026, by Selleck Chemicals (1 mentions)
Sb203580, by Cell Signaling Technology (1 mentions)
F ab 2 fragment alexa fluor 488 conjugate, by Cell Signaling Technology (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Actin antibody, by Merck Group (1 mentions)
Doxycycline, by AppliChem (1 mentions)
Anti ki67, by Agilent Technologies (1 mentions)
Rsk inhibitor lji308, by Selleck Chemicals (1 mentions)
Anti phospho histone h2a x ser139 antibody, by Merck Group (1 mentions)
Akt inhibitor mk 2206, by Selleck Chemicals (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
26 protocols using human phospho mapk array kit
1
Identifying Phosphorylated Proteins by MAPK Array
2
Western Blot Analysis of Cellular Signaling
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Cell lysates were extracted with CETi protein extraction solution (TransLab, Daejeon, Korea). Thirty to fifty micrograms of protein extract was used for western blotting with antibodies against p-Rbs, p21, cyclin D1, GSK3β, p-GSK3β, CatB, GS, p-GS, p38MAPK, p-p38MAPK, p70S6K, p-p70S6K (Cell Signaling, Denvers, MA, USA), α-tubulin, mouse IgG peroxidase conjugate, rabbit IgG peroxidase conjugate (Sigma-Aldrich Corp.) and Rb (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The human phospho-MAPK Array Kit (R&D Systems, Inc., Minneapolis, MN, USA) was used according to the manufacturer's instructions. A polyclonal antibody against CST1 was obtained from mice immunized with the purified protein.
3
Endothelial Cell Response to Cancer EVs
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To explore the intracellular signalling mechanisms triggered in HUVEC or HBEC-5i cells by cancer EVs, 30 μg/mL of GSC-derived EVs were combined with endothelial cells and incubated for six days. The cells were collected, lysed in RIPA buffer and the relative expression of phosphorylated kinases was analysed using Human Phospho-RTK Array kit and the Human Phospho-MAPK Array kit (both from R&D Systems, Minneapolis, MN, USA), followed by quantification using Fiji software.
4
Quantifying Cellular Kinase Phosphorylation
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The Human Phospho‐MAPK Array Kit (ARY003B, R&D Systems®, Inc. USA) was performed to detect the relative phosphorylation levels of 43 human kinases. Chemiluminescence Gel Imaging System (Bio‐Rad, USA) was used to detect the array's chemiluminescence signal (details of the steps are in Method S1 ).
5
Profiling MAPK Phosphorylation in Cancer Cells
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HCC827‐GRS and HCC4006‐GRS cells were treated with trametinib (100 nmol/L) plus taselisib (100 nmol/L). After 48‐hour treatment, the total protein was purified, and the cell lysates were applied to the Human Phospho‐MAPK Array Kit (R&D Systems, Minneapolis, MN, USA). The relative phosphorylation levels of 26 different MAPKs and serine/threonine kinases were determined in accordance with the manufacturer's instructions.
6
Phospho-MAPK Array Analysis of B7-H1 Signaling
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Human Phospho-MAPK Array Kit (R&D Systems) was used to screen phosphorylated proteins between B7-H1 and Mock transfected 624mel cells. After incubation, cells are fixed in 4% formaldehyde for 10 min at 37 °C followed with permeabilization in ice-cold methanol for 30 min on ice. After washing, cells were blocked with Fc receptor blockers and incubated with rabbit mAb to phospho-ERK1/2 (T202/Y204) (clone 197G2) or p38 MAPK (Thr180/Tyr182) (Clone D3F9, Cell Signaling #4511) or Isotype control IgG for 1 hour at room temperature, followed with incubation with secondary anti-rabbit IgG (H + L), F (ab')2 Fragment (Alexa Fluor® 488 Conjugate) (Cell Signaling #4412) for 30 min at room temperature. To inhibit the activity of DNA-PKcs, NU7026 (Selleckchem #S2893) was added at 1 or 10 μM in cell culture an hour before analysis. To inhibit the kinase activity of p38 MAPK, SB203580 (Cell Signaling #5633) was added at 10 μM in cell culture. The intracellular levels of ERK or p38 MAPK activation was analyzed by flow cytometry.
7
Quantitative Analysis of Phospho-MAPK Signaling
8
Western Blot Analysis of Key Signaling Proteins
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The antibodies for Western blot analyses of P-YB-1 (S102) (#2900), YB-1 (#4202), P-Akt (S473) (#4060), GAPDH (#2118S), cleaved PARP (#9541S), P-p53 (S20) (#9287P), p53 (#9282S) and p21 (#2947S) were purchased from Cell Signaling Technology (Frankfurt, Germany). The Akt antibody (#610877) was purchased from BD Biosciences (Heidelberg, Germany). The KRAS antibody (#ab157255) and the antibody for immunofluorescence staining of P-YB-1 (#ab47162) were purchased from Abcam (Cambridge, UK). The actin antibody (#A2066) was purchased from Sigma-Aldrich (Taufkirchen, Germany). The anti α-tubulin antibody (cat. #CP06) was purchased from Calbiochem (Schwalbach, Germany). Anti-Ki67 (#A2066) was purchased from Agilent (Waldbronn, Germany). The Human Phospho-MAPK array kit was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Anti-phospho-Histone H2AX (Ser139) antibody (#05-636) was purchased from Merck Millipore (Darmstadt, Germany). The RSK inhibitor LJI308 (#S7871) and Akt inhibitor MK2206 (#S1078) were purchased from Selleckchem (Munich, Germany). 5-fluorouracil (#84300-VO) was purchased from Medac (Hamburg, Germany). Cetuximab was provided by the pharmacy at the University of Tuebingen (Tuebingen, Germany). Doxycycline (#A2951) was purchased from AppliChem (Darmstadt, Germany).
9
Phospho-kinase Profiling in Cancer Cells
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Phospho-proteome profiler array has been performed by following manufacturer’s protocol (Human Phospho-MAPK Array Kit, R&D Systems, Minneapolis, MN, USA). Briefly, GEO-CR cancer cells were seeded in 6-well plates, treated with cetuximab (2.5 μg/mL) and 4-IPP (25 μM) for 24 h, rinsed with PBS and solubilized with Lysis Buffer 6 (provided in Human Phospo-MAPK Array) at 1 × 107 cells/mL. Samples were rocked for 30 min at 4 °C, centrifuged for 5 min at 14,000× g at 4 °C and the supernatants were collected and subjected to protein content determination by using the BioRad Reagent (BioRad, Milan, Italy). Protein lysates (300 μg) were incubated overnight with human phospho-kinase array in order to analyze the phosphorylation profile of the following 26 kinases: Akt1, Akt2, Akt3, Akt pan, CREB, ERK1, ERK2, GSK-3α/β, GSK-3β, HSP27, JNK1, JNK2, JNK3, JNK pan, MEK3, MEK6, MSK2, p38α, p38β, p38δ, p38γ, p53, p70 S6 Kinase, RSK1, RSK2, TOR. The levels of phosphorylated proteins were analyzed by quantification of the pixel density of each spot on the array by using the ImageLab software (BioRad), according to the manufacturer’s recommendations.
10
Comprehensive Phospho-Kinase Profiling
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The activation status of 42 RTKs and 26 protein kinases including 9 MAPKs was assessed using the Proteome Profiler human phospho-RTK (pRTK) array kit and human phospho-MAPK array kit (R&D Systems, Minneapolis, MN), as previously described [23] (link).
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