Figure 1: Schematic representation of the position of targets in a single well.
Figure 2: NIH/3T3 Cells were starved and stimulated with PDGF (100 ng/ml) for 5 minutes or IL-6 (100 ng/ml) for 5 minutes at 37°C. For UV treatment, exponentially growing NIH/3T3 cells were irradiated without serum starvation. Lysates were prepared and analyzed using the PathScan® Cell-Growth 4-Plex Array Kit. Luminescent signals were acquired using a Quansys Biosciences imager. Spot intensity was quantified using Q-View™ software. The bar graph shows measurements of four different target proteins. Corresponding Western blots obtained with the same cell lysates are shown at the bottom.
Figure 3: Cell extracts from unstimulated or stimulated NIH/3T3 cells were applied to wells at increasing total protein concentrations and analyzed. Luminescent signals were acquired and spot intensity quantified. The graphs show the relationship between the total protein concentration and the resulting strength of the luminescent signal for each target.
CST’s PathScan® Inflammation 4-Plex Array Kit is based upon the sandwich ELISA principle. Rather than one immobilized antibody, these PathScan® 4-Plex Array Kits contain four different antibodies in each well of a 96-well plate. This allows the researcher to measure four different target proteins from a single well simultaneously. In addition, the incorporation of multiple phospho-specific antibody pairs allows the researcher to measure the activation state of important signaling nodes. The PathScan® Inflammation 4-Plex Array Kit allows the detection of: Phospho-p38α MAPK (Thr180/Tyr182); Phospho-Stat3 (Tyr705); Phospho-p44 MAPK (Thr202/Tyr204); and Total Akt1 protein.
Briefly, 4 capture antibodies with distinct target specificity have been spotted onto the bottom of each well of a microwell plate. After incubation with cell lysates, the spotted antibodies capture the target proteins. Following extensive washing, a mixture of detection antibodies is added to detect the captured target proteins. An HRP-linked secondary solution is then used to recognize the bound detection antibodies. Chemiluminescent HRP substrate is used to produce luminescent signal.
Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).
PathScan® Inflammation 4-plex Array Kit detects endogenous levels of all target proteins. As shown in Figure 2, stimulation of NIH/3T3 cells with PDGF promotes phosphorylation of p44 MAPK at Thr202/Tyr204 and a modest induction of Stat3 phosphorylation onTyr705, while p38 MAP kinase remains unaffected. Stimulation with IL-6 or UV induces phosphorylation of Stat3 or p38, respectively, while not affecting measured phosphorylation levels of the other targets. Levels of total Akt1 remain essentially unchanged with all treatments.
Cells must respond in an appropriate fashion to many complex signaling events. Extracellular signaling cues are organized into well defined signal transduction modules that control fundamental cellular behvior. Three prominent signaling modules that are among the best characterized are the p44/42 MAP kinase (ERK MAPK), p38 Map kinase and the JAK-Stat signal transduction pathways. p44/42 MAPK is activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. p44/42 activation occurs through phosphorylation of threonine and tyrosine at the sequence T*EY* by a dual specificity kinase called MAP kinase kinase (MEK). p38 MAPK is dually phosphorylated in response to pro-inflammatory cytokines or cellular stress. Stat3 (Signal Transducers and Activators of Transcription) is phosphorylated at Tyr705 in response to a variety of cytokines and growth factors. Upon phosphorylation, Stat3 translocates to the nucleus where it acts as a transcription factor. Phosphorylation levels of critical molecular switches such as MAPKs therefore serve as a reliable indicator of the activation state of the entire signaling module. The profiling of phosphorylation events using phospho-specific antibodies is now widely used to investigate diagnostic pathology (1,2). While profiling of protein phosphorylation events was shown to predict the progression of a tumor to a more invasive stage (3), it has been observed that the ratio between p44/42 and p38 MAPK may predict whether tumor cells will proliferate or enter a dormant state in vivo (4). PathScan® Inflammation 4-Plex Array Kit provides the researcher with means to profile numerous chemical compounds and obtain in-cell relative potency (5).
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