Name: Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET (Video) | JoVE Journal
Uploaded: 2012-01-27T12:00:00.000+00:00
Duration: 13 min 38 s
Description: Learn to monitor kinase and phosphatase activities through the cell cycle using ratiometric FRET in live U2 OS cells. This method enables real-time visualization of phosphorylation changes during G2 to M transition.

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5 minutes

The authors are supported by the Swedish research council, the Swedish foundation for strategic research, the Swedish cancer society, the Swedish child cancer society, &Aring;ke Wibergs foundation and Jeanssons foundation.

1. Introducing the probe to cells
<ol>
 <li>Co-transfect cells with a FRET-based probe and a plasmid conferring resistance. Choose a transfection method that is efficient in your cell-type of interest. For U2OS cells, standard calcium phosphate transfection methods give adequate results15.</li>
 <li>Select cells in the appropriate antibiotic for at least seven days. This enriches the amount of cells expressing the probe and limits the amount of cells with toxic expression levels, or expression levels that severely affect the cell cycle.</li>
 <li>(optional) Select for stable clones.</li>
 <li>Using fixed or live cells, verify that both CFP and YFP are present in all cells at approximately the same ratio.</li>
 <li>In case some cells contain only CFP or YFP, recombination has likely occurred. Recombination can occur either during bacterial growth or after transfection of the probe and may in the latter case depend on plasmid quality. Repeat plasmid preparation and linearize the plasmid before transfection if the problem persists.</li>
</ol>
2. Monitoring ratiometric FRET
<ol>
 <li>Seed cells on glass bottom dishes, or cover slips for mounting in a chamber. Make sure that dishes/cover slips are no 1.5 (170 μm thick).</li>
 <li>Mount cells in growth medium without phenol red on a motorized epifluorescence microscope with temperature control set to 37 °C. Ensure pH regulation of media, either by CO2 or by using CO2-independent media such as Liebowitz-15.</li>
 <li>Focus on cells using transmitted light. Do not look on cells using fluorescent light.</li>
 <li>Start by acquiring a 12 or 16 bit image using YFP excitation (YFPex) and YFP emission (YFPem) filters and a suitable dichroic mirror. Use maximal binning available that still permits the required subcellular resolution. Using a high binning to decrease exposure time or intensity is crucial to avoid phototoxicity.</li>
 <li>Measure or estimate the background intensity and the approximate noise by measuring or estimating the rough average, minimum and maximum pixel values in an area devoid of cells. Change exposure time and neutral density filters so that the average signal in the cell is approximately the background intensity plus five to ten times the difference between max and min background intensities.</li>
 <li>Verify that images are not close to being saturated. The maximum intensity in the image should be less than half of the dynamic range (e.g. max 2000 in a 12 bit image) to allow for intensity differences when cells enter mitosis.</li>
 <li>Repeat step 2.4 to 2.6 using CFP excitation and YFP emission filters and a suitable dichroic mirror.</li>
 <li>Select multiple regions with transfected cells, excluding the regions used for point 2.4 to 2.7, and ensure that maximum intensity is less than half of the dynamic range in all cells.</li>
 <li>Start a time-lapse experiment acquiring two images every 45 min using YFPex - YFPem and CFPex - YFPem filter sets.</li>
</ol>
3. Verifying and optimizing conditions
<ol>
 <li>Open the acquired images and monitor the cell cycle time from mitosis to mitosis for cells that express the transfected probe.</li>
 <li>Compare the measured cell cycle time to published data for the cell type used. Alternatively, film untransfected cells by acquiring one image of transmitted light only (e.g. DIC or phase-contrast) every hour to get reference cell cycle times.</li>
 <li>In case the time from mitosis to mitosis corresponds to reference cell cycle timings, proceed to step 4. Alternatively, re-acquire images using harder exposure, more time-points or multiple z-levels and repeat step 3.</li>
 <li>In case the time from mitosis to mitosis does not correspond to reference cell cycle timings, film transfected cells by acquiring one image of transmitted light only (e.g. DIC or phase-contrast) every hour followed by one image at the end of the experiment to identify transfected cells.</li>
 <li>If the transfected cells still show longer cell cycle times, repeat step 1 but transfect less probe or select for stable clones that contain very low amounts of probe.</li>
 <li>If the transfected cells show normal cell cycle times in the absence of fluorescent light, repeat step 2 but lower exposure by modifying binning, neutral density filters, exposure time, and time between images.</li>
</ol>
4. Analysing FRET
<ol>
 <li>The analysis can be performed by most software dedicated to microscopy. Here we describe how to perform the analysis using the freeware ImageJ (<a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a>) utilizing the plugin Ratio Plus (<a href="http://imagej.nih.gov/ij/plugins/ratio-plus.html" target="_blank">http://imagej.nih.gov/ij/plugins/ratio-plus.html</a>).</li>
 <li>Open the images in ImageJ. In case your images are in the format of a multicolor stack (e.g. Deltavision), separate the individual channels by first converting them to a hyperstack: Image-hyperstack-stack to hyperstack and subsequently split them to single channel stacks using: Image-hyperstack-reduce dimensionality</li>
 <li>Multiply the YFPex - YFPem stack with 3 using: Process-Math-Multiply This step is optional, as it is only intended to increase the visual clarity of the images by ensuring that ratios are not in the range between 0 and 1.</li>
 <li>In the YFPex - YFPem stack, draw a region of interest (ROI) in an area that is devoid of cells, but that is close to a cell to be measured. Different cells in the same image may require different regions, as both background and signal intensity typically is strongest in the center of an image.</li>
 <li>Add the ROI to ROI manager: Analyze-tools-ROI manager.</li>
 <li>Measure the average intensity of the ROI in both the YFPex - YFPem and the CFPex - YFPem stack: Analyze-Measure.</li>
 <li>Repeat at multiple timepoints and verify that background intensities close to the cell of interest are similar throughout the film.</li>
 <li>Set measurements to include minimal intensity: Analyze-Set measurements-Min and Max grey value.</li>
 <li>Draw a region of interest covering most of a cell and measure the minimal intensity in both the YFPex - YFPem and the CFPex - YFPem stack. Take the difference between the measured minimal intensity and the background intensity from 4.6 and divide it by two. This provides a starting estimate for a clipping value.</li>
 <li>Open Ratio Plus. Select the YFPex - YFPem and the CFPex - YFPem stack. For FRET ratio, select CFPex - YFPem as stack 1, for inverted FRET ratio visualizing probes where FRET efficiency decreases upon phosphorylation, select YFPex - YFPem as stack 1.</li>
 <li>Insert the measured background intensities and clipping values.</li>
 <li>Set the scaling of the resulting ratio stack and apply a suitable look up table (LUT) to visualize ratio changes.</li>
</ol>
5. Representative Results
Plk1 activity is first visible in the nucleus in G2 phase and peaks during mitosis. Figure 3 shows an experiment using minimal phototoxicity settings as described in section 2. Please note that this is a representative initial result and that exposure conditions or time between images can be modified to increase signal to noise ratio or temporal resolution. Figure 3D shows that a majority of the cells expressing the probe proliferate with a cell cycle time of between 20 and 25 hours, indicating that the imaging conditions and expression levels of the probe do not affect cell cycle timings. Although there is considerable noise, the trend of Plk1 activity increasing in G2 and peaking in mitosis14 is clearly visible (fig 3A). Processing the raw data, here by mean filtering presented as a kymograph (fig 3B), or quantification of the average inverted ratio (fig 3C) can enhance clarity.
<img alt="Kinase-phosphatase cycle diagram, protein regulation, enzyme action, phosphorylation process." src="/files/ftp_upload/3410/3410fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. Principle of a FRET-based probe to monitor kinase and phosphatase activities. Two fluorophores, typically CFP (blue) and YFP (green), are connected by a phospho-binding domain (orange) and a phosphorylatable sequence (yellow). Phosphorylation (red) mediates binding to the phospho-binding domain, thereby inducing a conformational change in the probe. The conformational change results in a difference in the distance or orientation between the two fluorophores, which affects the FRET efficiency between CFP and YFP. FRET can be visualized by exciting CFP and monitoring YFP emission (dotted lines).
<img alt="Plasmid transfection process diagram, epifluorescence microscopy, FRET ratio analysis steps." src="/files/ftp_upload/3410/3410fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2. Schematic outline of the experimental procedure.
<img alt="Time-lapse imaging and graphs of protein expression and mitotic entry, fluorescence data analysis." src="/files/ftp_upload/3410/3410fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3. Plk1 activity is first visible in the nucleus in G2 phase and peaks during mitosis. U2OS cells expressing a FRET-based probe monitoring Plk1 activity9,14 were filmed for 60 hr using a Deltavision Spectris Imaging system equipped with a 20x NA 0.7 air objective and a mercury lamp. Imaging conditions were selected to cause minimal phototoxicity, as outlined in section 2, using 4x binning and neutral density filters that block 99% of the incoming light. A, false color representation of inverted FRET-ratio, following one cell through 4 divisions. B, kymograph of the cell shown in A after applying a mean filter. C, quantification of the inverted FRET ratio of the cell shown in A. D, cumulative mitotic entry of 50 FRET-probe expressing cells, including divisions of daughter cells.

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<li>Fosbrink, M., Aye-Han, N. N., Cheong, R., Levchenko, A., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+JNK+activity+dynamics+with+a+genetically+encoded+fluorescent+biosensor%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5459-5464 (2010).</li>
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<li>Morgan, D. O. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMorgan%2C+DO+%22The+cell+cycle+%3A+principles+of+control%22">The cell cycle : principles of control</a>. , New Science Press, in association with Oxford University Press. (2007).</li>
<li>Lindqvist, A., Rodriguez-Bravo, V., Medema, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+decision+to+enter+mitosis%3A+feedback+and+redundancy+in+the+mitotic+entry+network%5BTitle%5D)+AND+%22J.+Cell.+Biol%22%5BJournal%5D)">The decision to enter mitosis: feedback and redundancy in the mitotic entry network.</a> J. Cell. Biol. 185, 193-202 (2009).</li>
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<li>Macurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polo-like+kinase-1+is+activated+by+aurora+A+to+promote+checkpoint+recovery%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.</a> Nature. 455, 119-123 (2008).</li>
<li>van der Eb, A. J., Graham, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assay+of+transforming+activity+of+tumor+virus+DNA%5BTitle%5D)+AND+%22Methods.+Enzymol%22%5BJournal%5D)">Assay of transforming activity of tumor virus DNA.</a> Methods. Enzymol. 65, 826-839 (1980).</li>
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We have nothing to disclose.

Monitoring FRET throughout the cell cycle requires considerations that are less crucial when assessing short-term responses to external stimuli. First, cell cycle progression is easily perturbed by stress signaling, requiring that phototoxicity is kept to a minimum. Second, all reporters can potentially affect cellular processes by titrating out kinases, phosphatases or interaction domains. The probably most straightforward way to assess if the experimental conditions are adequate is to measure the cell cycle length from...

<table><tbody><tr><td>Leibovitz L-15, no phenol red</td><td>GIBCO, by Life Technologies</td><td>21083-027</td><td></td></tr><tr><td>DMEM+Glutamax-I</td><td>GIBCO, by Life Technologies</td><td>31966</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) </td><td>Hyclone</td><td>SV30160.03</td><td></td></tr><tr><td>0.05% Trypsin EDTA </td><td>Hyclone</td><td>SH30236.01</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>DPBS </td><td>GIBCO, by Life Technologies</td><td>14287</td><td></td></tr><tr><td> Puromycin </td><td>Sigma-Aldrich</td><td>P8833</td><td></td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

In this video, kinase and phosphatase activities are monitored throughout the cell cycle using Forrester Resonance Energy transfer or fret. Here a polo like kinase one or LK one activity probe has cyan fluorescent protein CFP placed on one end and yellow fluorescent protein YFP placed on the other. This probe is transfected into U2 OS cells.Then to examine the phosphorylation status of LK one targets during the G two two M transition, the TRANSFECTED U2 OS cells are subjected to time lapse fluorescence imaging. When CFP and YFP are in close proximity and CFP is excited, energy is transferred to YFP and YFP admission can be detected. However, when the probe becomes phosphorylated, a confirmational change in the protein separates CFP and YFP.Now when CFP is excited, a strong CFP emission and only a weak YFP emission are detected quantification of the ratio of YFP emission after both CFP and YFP excitation demonstrates that PLK one activity increases in G two and peaks during mitosis. So the main advantage of this technique over existing methods like standard geometric filming, is that specific attention is put to that the cell cycle is not perturbed either by filming conditions or by expression of a probe. This method can help answer key questions in the cell cycle field, such as where and when specific kinases or phosphatases are activated.Although this method can provide insight into cell cycle regulated enzymes, it can also be applied to other systems where unres conditions are required, such as cell proliferation assays. To monitor the human cell cycle, U2 OS cells are transfected with a fret based PK one activity probe. Using a standard calcium phosphate transfection method, the cells are selected in pur mycin for at least seven days.This enriches the population of cells expressing the probe and limits the number of cells with toxic expression levels or expression levels that severely affect the cell cycle. Stable clones are then selected by limited dilution expression of CFP and YFP is then examined to verify that they are present in all cells at approximately the same ratio. If some cells contain only CFP or YFP recombination has likely occurred in this case, repeat the plasmid preparation using bacteria that are less prone to homologous recombination, such as sure two cells then linearize the plasmid and repeat the transfection.Once the cells stably express the probe, seed them on number 1.5 glass bottom dishes after they have attached and are 30 to 50%confluent. Change the medium to one that is CO2 independent and does not contain phenol red mount the cells on a motorized epi fluorescence microscope With temperature control set to 37 degrees centigrade. The microscope should be equipped with CFP and YFP excitation and emission filters.A ROIC mirror suitable for monitoring CFP and YFP and an objective suitable for the required resolution. Here, a 20 x NA 0.75 air objective is used. Using transmitted light, bring the cells into focus.Do not use fluorescent light at this point since it will induce a stress response that may perturb the cell cycle. To avoid phototoxicity, set the bending to the maximum available that permits the required subcellular resolution. For this experiment, it should be possible to distinguish the nucleus and the cytoplasm.Set a short exposure time and insert a high neutral density filter. Then acquire a 12 or 16 bit test image Using YFP excitation and emission, adjust the exposure time and neutral density filters so that the average intensity in a cell is approximately equal to the background intensity plus five times the difference between minimum and maximum background intensities. To verify that the images are not saturated, examine the fluorescence intensity for the sample image.The maximum intensity in the image should be less than half of the dynamic range to allow for intensity differences when cells enter mitosis. Next, repeat this process using CFP excitation and YFP emission filters. Select multiple regions with transfected cells, excluding the regions used for focusing and optimizing exposure conditions.Since these procedures may have invoked a stress response, ensure that the maximum intensity is less than half of the dynamic range in all of the cells. Instruct the software to acquire two images every 45 minutes using YFP excitation, YFP emission and CFP excitation. YFP emission filters.Also set the total length of the experiment to exceed the duration of the cell cycle. Once everything is set, begin acquiring images at after the acquisition is complete, open the acquired images and monitor the cell cycle time from mitosis to mitosis. For cells that express the transfected probe here, the cell cycle time was roughly 20 to 25 hours.Compare this to control data for the cell type used. If cell cycle time corresponds to reference timings fret can be analyzed. Once the timing has been verified, the images can be analyzed.The analysis can be performed by most software dedicated to microscopy. Here, image j freeware is used with the ratio plus plugin. Begin the analysis by opening the images in Image J.If the images are in a multicolor stack format, separate the individual channels by converting them to a hyper stack By selecting image.Then hyper stack then stack to hyper stack. The convert to hyper stack window opens with the settings displayed click okay. The image will now be displayed with two slider bars below it.Next to split the images to single channel stacks, click on image and select hyper stack reduce dimensionality. This will open the reduce window, ensure that frames and keep source are selected. Then click Okay.A new image containing only one channel will appear. Repeat this process to bring up a new image with the second channel. Next, to increase the visual clarity of the final images, select the YFP excitation YFP emission stack.Click on process, then click on math. Then multiply When the multiply window opens, enter three to multiply the YFP excitation YFP emission stack by three. The step is optional and ensures that ratios are not in the range between zero and one.Next, on the image J toolbar, select the freehand tool. Then in the YFP excitation, YFP emission stack, draw a region of interest or ROI in an area that is devoid of cells, but that is close to a cell that will be measured to add the ROI to the ROI manager, click on analyze tools. Then ROI manager.The ROI manager window will appear. Click on add to save the ROI coordinates to measure the average intensity of the ROI In the YFP excitation YFP emission stack. Click on analyze, then select measure.The results window will open. Select the CFP excitation YFP emission stack. Then double click on the ROI in the ROI manager and repeat the measurement.The measurement is added to the results window. Scroll to other places in the stack to repeat this process at multiple time points. Verify that background intensities close to the cell of interest are similar throughout the film by comparing measurements in the results window.To set measurements to include minimal intensity, click on analyze, then click on set measurements and indicate the minimum and maximum gray value. A window showing measurement alternatives will appear next. After adjusting brightness and contrast as necessary, draw an ROI covering most of a cell and measure the minimal intensity in both stacks.Then take measurements as before this time using a cell instead of the background. Using the values from the results sections, calculate the difference between the measured minimal intensity and the background intensity and divide it by two. This provides a starting estimate for a clipping value to open ratio plus select plugins.Then ratio plus. Then for fret ratio, select the CFP excitation YFP emission S stack one or for inverted fret ratio visualizing probes where fret efficiency decreases upon phosphorylation. Select the YFP excitation YFP emission S stack one to insert the measured background intensities and clipping values.Use the values from the result window and paste them in the ratio plus window. Set the scaling of the resulting ratio stack and apply a suitable lookup table to visualize ratio changes. U2 OS cells expressing a fret based probe monitoring PLK one activity were filmed for 60 hours as described in this video.This image shows cumulative mitotic entry of 50 fret probe expressing cells, including divisions of daughter cells. The majority of the cells expressing the probe proliferate with a cell cycle time of between 20 and 25 hours, indicating that the imaging conditions and expression levels of the probe do not affect cell cycle timing. A false color representation of inverted fret ratio following one cell through four divisions demonstrates that although there is considerable noise, a trend is seen in which PK one activity increases in G two and peaks during mitosis.When the raw data is mean filtered and presented as a graph, the time between activity peaks is easy to estimate. This figure shows quantification of the inverted fret ratio of the cell shown in the previous images from the graph. The timing of activation can be estimated and as can be seen here, phosphorylation of the probe becomes visible approximately four hours before the phosphorylation is peaking in mitosis.While attempting this procedure, it's important to keep the cells in an unstress condition, so this is done by minimizing exposure to light as well as to minimize changes in temperature or pH. It is also important to remember that this method monitors the balance between kinase and phosphatase activity in a cell. This procedure can be combined with RNA interference or inhibitor screens to identify the upstream regulators of these kinases and phosphotases.

monitoring-kinase-phosphatase-activities-through-cell-cycle

Research

JoVE Journal

Biology

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol.  We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analysis of Cell Cycle Position in Mammalian Cells

Förster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

This video describes the time-resolved Förster resonance energy transfer or TR-FRET assay to determine endogenous protein phosphorylation with the help of donor and acceptor fluorophores tagged on antibodies specific to a test protein. This technique is designed to measure the phosphorylation of test proteins in cell lysates.

Time-Resolved Forster Resonance Energy Transfer for Monitoring Protein Phosphorylation in Cells

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([γ-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Assaying Protein Kinase Activity with Radiolabeled ATP

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Time-resolved Förster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

The protocol presented in this study illustrates the effectiveness of the cAMP Difference Detector In Situ  in measuring cAMP through two methods. One method utilizes a 96-well plate reading spectrophotometer with HEK-293 cells. The other method demonstrates individual HASM cells under a fluorescent microscope.

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ

Here, we present a protocol to quantify the spatiotemporal dynamics of Akt activation and phosphorylation in live HepG2 cells. Förster resonance energy transfer (FRET) imaging is a powerful tool that provides valuable insights into insulin signaling pathways and metabolic regulation in cancer cells.

Live-Cell F&#246;rster Resonance Energy Transfer Imaging of Metabolically Regulated Akt Activation Dynamics in HepG2 Cells

Learn to monitor kinase and phosphatase activities through the cell cycle using ratiometric FRET in live U2 OS cells. This method enables real-time visualization of phosphorylation changes during G2 to M transition.

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

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