For the simultaneous detection of 19 signaling molecules that are involved in the regulation of the stress response and apoptosis, the PathScan® Stress and Apoptosis Signaling Antibody Array Kit has been used according to the manufacturer’s instructions (Cell Signaling Technology, Danvers, MA, USA). Briefly, MCF-7 cells were grown until 80% confluence, treated with the selected compounds, and lysed in 1X Cell Lysis Buffer to collect cell lysates. The array-blocking buffer was added to each well for 15 min at room temperature. Then, 30 μg of solubilized proteins were added to wells and incubated for 2 h at room temperature. Subsequently, the Detection Antibody Cocktail supplied with the kit was added and maintained for 1 h at room temperature. The slide was then incubated for 30 min with horseradish peroxidase-linked streptavidin solution at room temperature. Finally, the slide was covered with LumiGLO/Peroxide reagent (supplied with the kit) and exposed to chemiluminescence film (Amersham Biosciences, Little Chalfont, UK) for 2 to 60 sec. The images were then acquired and the signal intensity was measured using the ImageJ software for Microsoft Windows (National Institutes of Health, Bethesda, MD, USA).
Pathscan stress and apoptosis signaling antibody array kit
Manufactured by Cell Signaling Technology
Sourced in United States
The PathScan® Stress and Apoptosis Signaling Antibody Array Kit is a slide-based antibody array designed to simultaneously detect the relative levels of 19 significant proteins involved in stress and apoptosis signaling pathways. The kit utilizes the sandwich immunoassay principle to detect and quantify the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Chemiluminescence film, by Cytiva (1 mentions)
Chemidoc xrs chemiluminescence detection and imaging system, by Bio-Rad (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Bca protein assay kit, by Sangon (1 mentions)
Pathscan intracellular signaling array kit, by Cell Signaling Technology (1 mentions)
Pathscan sandwich elisa lysis buffer, by Cell Signaling Technology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Imaging system, by LI COR (1 mentions)
Pathscan rtk signaling antibody array kit, by Cell Signaling Technology (1 mentions)
Peroxide lumiglo reagent, by Cell Signaling Technology (1 mentions)
Sk mel 28, by Korean Cell Line Bank (1 mentions)
Minimum essential medium (mem), by Welgene (1 mentions)
Sk mel 2, by Korean Cell Line Bank (1 mentions)
Sk mel 2, by Welgene (1 mentions)
G 361, by Welgene (1 mentions)
Wm 266 4, by Korean Cell Line Bank (1 mentions)
Sk mel 5, by Korean Cell Line Bank (1 mentions)
18 protocols using pathscan stress and apoptosis signaling antibody array kit
1
Signaling Pathways: Stress and Apoptosis
2
Multiplex Signaling Profiling in Tumors
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Eight tumor samples were randomly selected from each group for all the following molecular analyses in tumors. A slide-based antibody array was used for simultaneous detection of 19 important signaling molecules involved in stress response and apoptosis, using a PathScan Stress and Apoptosis Signaling Antibody Array kit (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions. Each of these molecules was detected in duplicate on the same array arranged as shown in Fig. 2c , and the identity of each numbered molecule listed in Fig. 2d . Briefly, total protein was extracted from tumor tissues using RIPA buffer (Santa Cruz Technology, CA), and diluted to 0.5 mg/mL in Array Diluent Buffer provided by the kit. The protein samples were incubated with the array antibodies overnight. A Detection Antibody Cocktail was added to the samples, followed by the addition of HRP-linked Streptavidin and substrate. Protein was visualized using a ChemiDoc XRS chemiluminescence detection and imaging system (Bio-Rad Laboratories, Irvine, CA). The density of the spots were quantitated using Quantity One software (Bio-Rad Laboratories) and normalized to the α-tubulin levels. Each sample was done in duplicate.
3
Comprehensive Stress and Apoptosis Signaling
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Cell extracts were prepared and analyzed using the PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) #12856, Cell Signaling Technology, MA, USA. Assay target proteins were P44/42 MAPK (ERK1/2) phosphorylation, AKT phosphorylation, BAD phosphorylation, HSP27 phosphorylation, SMAD2 phosphorylation, p53 phosphorylation, p38 MAPK phosphorylation, SAPK/JNK phosphorylation, PARP cleavage, Caspase-3 cleavage, Caspase-7 cleavage, total Ikβα, Chk1 Ser345 phosphorylation, Chk2 phosphorylation, Ikβ α phosphorylation, eIF2α phosphorylation, TAK1 phosphorylation, Survivin and α-Tubulin as a reference protein.
Images were acquired by briefly exposing the slide to standard chemiluminescent film. Densitometry analysis was performer using ImageJ (http://imagej.nih.gov/ij/ ). Results are shown as a mean±SD normalized to the internal reference protein (α-Tubulin). Untreated negative control (UC) was set as 100% expression level.
Images were acquired by briefly exposing the slide to standard chemiluminescent film. Densitometry analysis was performer using ImageJ (
4
Stress and Apoptosis Signaling Assay
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The effect of BZD9L1 and/or 5-FU on HCT 116 stress and apoptosis signalling was
studied using the PathScan® Stress and Apoptosis Signaling Antibody
Array Kit (Cell Signaling Technology) according to the manufacturer’s protocol.
Briefly, the array gasket was fixed onto the array slide, and the array was
blocked with array blocking buffer for 15 min. Next, protein lysates (harvested
from cells 72 h post-treatment) were added into the gasket, sealed and allowed
to incubate at 4°C overnight. The gasket was subsequently washed with array
washing buffer (four times, 5 min each) followed by incubation with detection
antibody cocktail for 1 h at room temperature. The gasket was washed again with
array washing buffer (four times, 5 min each) and incubated with HRP-linked
streptavidin for 30 min at room temperature. At the end of the incubation
period, the gasket was washed again in array washing buffer as previously
described. The gasket was then removed and the array slide was briefly washed
further. The slide was exposed to the scanning reagent for 1 min and scanned
using a chemiluminescence imaging system (ChemiDoc XRS+, Bio-Rad, USA). The
intensity of array dots were analysed using Bersoft Array Analyzer software
(Bersoft Software and Technology, Canada).
studied using the PathScan® Stress and Apoptosis Signaling Antibody
Array Kit (Cell Signaling Technology) according to the manufacturer’s protocol.
Briefly, the array gasket was fixed onto the array slide, and the array was
blocked with array blocking buffer for 15 min. Next, protein lysates (harvested
from cells 72 h post-treatment) were added into the gasket, sealed and allowed
to incubate at 4°C overnight. The gasket was subsequently washed with array
washing buffer (four times, 5 min each) followed by incubation with detection
antibody cocktail for 1 h at room temperature. The gasket was washed again with
array washing buffer (four times, 5 min each) and incubated with HRP-linked
streptavidin for 30 min at room temperature. At the end of the incubation
period, the gasket was washed again in array washing buffer as previously
described. The gasket was then removed and the array slide was briefly washed
further. The slide was exposed to the scanning reagent for 1 min and scanned
using a chemiluminescence imaging system (ChemiDoc XRS+, Bio-Rad, USA). The
intensity of array dots were analysed using Bersoft Array Analyzer software
(Bersoft Software and Technology, Canada
5
Stress and Apoptosis Signaling Profiling
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Infected SMMC-7721 cells were collected and lysed. A PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Cell Signaling Technology, #7018) was then used, according to the manufacturer’s instructions,16 (link) to detect variations in intracellular signaling pathways.
6
Stress and Apoptosis Signaling Pathway Analysis
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PathScan analysis was performed using the PathScan® stress and apoptosis signaling antibody array kit (catalog no 12923; Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. Briefly, cells transfected with shEIF3C or shCtrl lentivirus were lysed with 1X Cell Lysis Buffer (catalog no 7018; Cell Signaling Technology). After collection of whole-cell lysates, protein concentration was determined using a BCA protein assay kit (Sangon Biotech). Proteins, at equal concentration, were analyzed by a PathScan sandwich ELISA kit according to the manufacturer’s instructions. The array target map used can be found at the manufacturer’s homepage (http://www.cst-c.com.cn/products/12856 . html). Slides were imaged using a ChemiScope5300 Pro Integrated chemiluminescence imaging system (CLiNX Science Instruments, Shanghai, China).
7
Intracellular Signaling Pathway Analysis
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Cell extracts were prepared and analyzed using the PathScan intracellular signaling array kit (Catalog no. 7323S; Cell Signaling Technology) and PathScan stress and apoptosis signaling antibody array kit (Catalog no. 12856S; Cell Signaling Technology) according to the manufacturer's instruction. The PathScan intracellular signaling array kit could simultaneously detect eighteen phosphorylated or cleaved intracellular signaling molecules including ERK1/2, mammalian target or rapamycin (mTOR), mitogen-activated protein kinase (MAPK), B-cell lymphoma-2-associated death domain (Bad). The PathScan Stress and Apoptosis Signaling Antibody Array could simultaneously detect nineteen apoptosis related signaling molecules including cleaved caspases (caspase 3 and 9) and PARP.
8
Stress and Apoptosis Signaling Assay
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Cells were lysed in Path Scan® Sandwich ELISA Lysis Buffer (Cell Signaling Technology, Danvers) and analyzed using PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Cell Signaling Technology, Danvers) according to the vendor's protocol. Chemiluminescent signals were detected via Odyssey Infrared Imaging System (application software version 3.0) from Li-Cor biosciences (Bad Homburg).
9
Signaling Molecules in TNF-sRII and Glucocorticoid-Induced Apoptosis
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To determine the signaling molecules that were involved in the regulation of TNF-sRII or glucocorticoids on the apoptosis of HA-SMCs in cellular interaction, the PathScan stress and apoptosis signaling antibody array kit (Cell Signaling Technology, 12923, Danvers, MA, USA) based upon the sandwich immunoassay principle was used. The array kit allowed for the simultaneous detection of 19 signaling molecules that were involved in the regulation of the stress response and apoptosis. The fluorescent image of the slide was captured with LI-COR imaging system, and spot intensities quantified using image studio software.
10
Simultaneous Signaling Pathway Analysis
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To simultaneously detect a wide range of vital and well-characterized signaling molecules, cell lysates were analyzed using the PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Cell Signaling Technology, #7982) and the PathScan® RTK Signaling Antibody Array Kit (Cell Signaling Technology, #12856) according to the manufacturer's instructions. After infection with shRAB10 or shCon for 5 days, SMMC-7721 cells were rinsed twice with ice-cold 1X phosphate-buffered saline (PBS), rapidly lysed in 1X cell lysis buffer and then incubated in Array Blocking Buffer for 20 min. An equal volume of lysate was placed in each sample and incubated for 2 h at room temperature. Before incubation with HRP-linked streptavidin, each reaction was incubated with detection antibody mixture for 1 h at room temperature. The slides were exposed to film for 25 sec after being developed with LumiGLO/Peroxide reagent (Cell Signaling Technology).
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