<meta charset="utf8"></head><body>Rapamycin-Regulated System을 이용한 인산가수분해효소 활성의 시간적 조절

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tyrosine+phosphatase+SHP2+regulates+focal+adhesion+kinase+to+promote+EGF-induced+lamellipodia+persistence+and+cell+migration.">The tyrosine phosphatase SHP2 regulates focal adhesion kinase to promote EGF-induced lamellipodia persistence and cell migration.</a> Mol Cancer Res. 11 (6), 651-664 (2013).</li><li>Yu, D. H., Qu, C. K., Henegariu, O., Lu, X., Feng, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-tyrosine+phosphatase+Shp-2+regulates+cell+spreading,+migration,+and+focal+adhesion.">Protein-tyrosine phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion.</a> J Biol Chem. 273 (33), 21125-21131 (1998).</li><li>Kramer, M. F., Coen, D. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#1.5 Glass Coverslips 25 mm Round</td>
			<td>Warner Instruments</td>
			<td>64-0715</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Tubes</td>
			<td>USA Scientific</td>
			<td>cc7682-3394</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Buffer</td>
			<td></td>
			<td></td>
			<td>For 500 mL: 5.18 g Tris-HCL, 131.5 mL glycerol, 52.5 mL 20% SDS, 0.5 g bromophenol blue, final pH 6.8</td>
		</tr>
		<tr>
			<td>4-20% Mini-PROTEAN TGX Precast Gel</td>
			<td>Biorad</td>
			<td>4561096</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Phusion Plus Buffer</td>
			<td>Thermo Scientific</td>
			<td>F538L</td>
			<td></td>
		</tr>
		<tr>
			<td>A431 Cells</td>
			<td>ATCC</td>
			<td>CRL-1555</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarsose</td>
			<td>GoldBiotech</td>
			<td>A-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Attofluor Cell Chamber</td>
			<td>invitrogen</td>
			<td>A7816</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchmark Fetal Bovine Serum (FBS)</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td>Heat Inactivated Triple 0.1 &#181;m sterile-filtered</td>
		</tr>
		<tr>
			<td>Brig 35,30 w/v %</td>
			<td>Acros</td>
			<td>329581000</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>GoldBiotech</td>
			<td>A-420</td>
			<td></td>
		</tr>
		<tr>
			<td>CellGeo</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Published in 10.1083/jcb.201306067</td>
		</tr>
		<tr>
			<td>CellMask Deep Red plasma membrane dye</td>
			<td>invitrogen</td>
			<td>c10046</td>
			<td></td>
		</tr>
		<tr>
			<td>Colony Screen MasterMix</td>
			<td>Genesee</td>
			<td>42-138</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5a competent cells</td>
			<td>NEB</td>
			<td>C2987H</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Thermo Scientific</td>
			<td>F-515</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Ladder</td>
			<td>GoldBio</td>
			<td>D010-500</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>NEB</td>
			<td>N04475</td>
			<td></td>
		</tr>
		<tr>
			<td>DpnI Enzyme</td>
			<td>NEB</td>
			<td>R01765</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>GoldBio</td>
			<td>DTT10</td>
			<td>DL-Dithiothreitol, Cleland&#39;s Reagents</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros</td>
			<td>409910250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE(R) 6 Transfection Reagent</td>
			<td>Promega</td>
			<td>E2692</td>
			<td>transfection reagent</td>
		</tr>
		<tr>
			<td>Gel extraction Kit</td>
			<td>Thermo Scientific</td>
			<td>K0692</td>
			<td>GeneJET Gel Extraction Kit</td>
		</tr>
		<tr>
			<td>Gel Green Nucleic Acid Stain</td>
			<td>GoldBio</td>
			<td>G-740-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Dye Purple 6x</td>
			<td>NEB</td>
			<td>B7024A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>GlutaMAX-l (100x) 100 mL</td>
		</tr>
		<tr>
			<td>HEK 293T Cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa Cells</td>
			<td>ATCC</td>
			<td>CRM-CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fischer</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Processing Software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA-630 (NP40)</td>
			<td>Sigma</td>
			<td>I3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole Buffer</td>
			<td></td>
			<td></td>
			<td>25 mM Imidazole pH 7.2, 2.5 mM EDTA, 50 mM NaCl, 5 mM DTT</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr>
			<td>L-15 1x</td>
			<td>Corning</td>
			<td>10-045-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher</td>
			<td>BP1425-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis Buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 1 mM EGTA, 1% NP40</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>N/A</td>
			<td>R 2022b update was used to run CellGeo functions</td>
		</tr>
		<tr>
			<td>Metamorph Microscopy Automation and Image Analysis Software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher Chemical</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma</td>
			<td>M5310</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniPrep Kit</td>
			<td>Gene Choice</td>
			<td>96-308</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Precast Gels 12 well</td>
			<td>Bio-Rad</td>
			<td>4561085</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water</td>
			<td>Corning</td>
			<td>46-000-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiband Polychroic Mirror</td>
			<td>Chroma Technology</td>
			<td>89903BS</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus UPlanSAPO 40x objective</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS w/o Ca and Mg</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Tubes</td>
			<td>labForce</td>
			<td>1149Z65</td>
			<td>0.2 mL 8-Strip Tubes and Caps, Rigid Strip Individually Attached Dome Caps</td>
		</tr>
		<tr>
			<td>Phusion Plus DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F630S</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protein-G Sepharose</td>
			<td>Millipore</td>
			<td>16-266</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Membranes</td>
			<td>BioRad</td>
			<td>1620219</td>
			<td>Immun-Blot PVDF/Filter Paper Sandwiches</td>
		</tr>
		<tr>
			<td>Rapamycin</td>
			<td>Fisher</td>
			<td>AAJ62473MF</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin, 2.21 mM EDTA, 1x [-] sodium</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Acetate-EDTA (TAE) 50x</td>
			<td>Fischer</td>
			<td>BP1332-1</td>
			<td>for electrophoresis</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 100 mM NaCl, 1 mM EGTA, 1% NP40</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Fisher Chemical</td>
			<td>O3446I-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-EGFR Antibody</td>
			<td>Cell Signaling</td>
			<td>2232</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Erk 1/2 Antibody</td>
			<td>Cell Signaling</td>
			<td>9102</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Flag Antibody</td>
			<td>Millipore-Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GAPDH Antibody</td>
			<td>invitrogen</td>
			<td>AM4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP Antibody</td>
			<td>Clontech</td>
			<td>632380</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mCherry Antibody</td>
			<td>invitrogen</td>
			<td>M11217</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-paxillin Antibody</td>
			<td>Thermo Fischer</td>
			<td>BDB612405</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-EGFR Y992 Antibody</td>
			<td>Cell Signaling</td>
			<td>2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-Erk 1/2 T202/Y204 Antibody</td>
			<td>Cell Signaling</td>
			<td>9101</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-paxillin Y31 Antibody</td>
			<td>Millipore-Sigma</td>
			<td>05-1143</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-PLC&#947; Y783 Antibody</td>
			<td>Cell Signaling</td>
			<td>14008</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PLC&#947; Antibody</td>
			<td>Cell Signaling</td>
			<td>5690</td>
			<td></td>
		</tr>
	</tbody>
</table>

development and application of rapamycin-regulated tyrosine phosphatases

Protein tyrosine phosphatases are a critical family of proteins involved in a plethora of cell signaling events. They have been shown to play a key role in the regulation of cell proliferation, migration, and apoptosis1,2,3. Consequently, the dysregulation of protein tyrosine phosphatases leads to a variety of debilitating diseases and disorders4,5,6,7. Studying the physiological function of tyrosine phosphatases and their role in the development of these pathologies has been historically hindered by a lack of tools needed for probing the intricacies of phosphatase signaling8.
Traditionally, phosphatases are studied using methods that do not have the desired specificity and/or do not provide temporal control of their activity. These critical limitations of available tools make it challenging to dissect specific roles of phosphatases in signaling pathways. Overexpression of constitutively active and dominant negative mutants or downregulation of expression of the phosphatase provide specificity but lack temporal control and often can trigger compensatory mechanisms that will mask the true function of the enzyme.
Pharmacological inhibitors allow for the temporal regulation of phosphatases. However, many phosphatase inhibitors are notoriously nonspecific due to the well-conserved composition of the active site in tyrosine phosphatases9. Additionally, due to design constraints, inhibitors targeting the catalytic site exhibit poor cell membrane permeability10. Another limitation of inhibitors is that they only allow for the examination of the effects of phosphatase inactivation11. Thus, there is a need for tools that enable specific, temporally regulatable activation of phosphatases. These tools will allow researchers to identify the direct effects of phosphatase activation, separating them from multiple parallel signaling cascades often activated by biological stimuli. Importantly, tight temporal control of activity enables the identification of transient events induced by a phosphatase and separates the effects of acute and prolonged phosphatase activity. Combining temporal regulation with mutational analysis will allow for a detailed dissection of specific roles of individual domains of the phosphatase and interrogation of its downstream signaling12.
To address the lack of desired capabilities in the existing tools, the Karginov group has developed the Rapamycin Regulated (RapR) system13,14,15. The RapR system utilizes an engineered switch domain, iFKBP, that allows for allosteric regulation of the protein of interest (POI). The insertion of the iFKBP domain at a position allosterically coupled to the catalytic site of the POI renders it susceptible to regulation by rapamycin. In the absence of rapamycin, iFKBP disrupts the catalytic site due to the intrinsically high structural dynamics of iFKBP and thus inactivates the POI. The addition of rapamycin induces the interaction of iFKBP with the co-expressed protein FRB (Figure 1). This causes stabilization of the switch domain, which consequently restores the structure and function of the POI&#39;s catalytic domain. As such, the tool allows for specific and temporally regulatable activation of the POI.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67142/67142fig01.jpg" /> 
Figure 1: Schematic of the RapR-Shp2 rapamycin-regulated system. RapR allows for allosteric activation of the protein of interest with the addition of rapamycin. This figure was modified from Fauser et al.12. Abbreviations: iFKBP = insertable FKBP12; FRB = FKBP-rapamycin-binding domain; R = rapamycin; Shp2 = Src homology-2 domain-containing protein tyrosine phosphatase. <a href="https://www.jove.com/files/ftp_upload/67142/67142fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The RapR tool can be applied to different protein families. It can be used to regulate protein kinases as well as phosphatases12,14. This protocol will focus on the application of the RapR tool to control Shp2 phosphatase. Shp2 is a ubiquitously expressed protein tyrosine phosphatase that is involved in signaling processes such as proliferation, migration, immunomodulation, and differentiation1,16,17,18. Dysregulation of Shp2 has been associated with a number of solid cancers, myeloid leukemia, and developmental disorders5,7. However, Shp2 has fallen victim to the same tool shortcomings as described above. To combat these limitations, RapR-Shp2, a specifically and temporally regulatable Shp2 construct, was developed and characterized12.
Prior to the development of RapR-Shp2, it was known that Shp2 was involved in cell migration19,20,21. However, its specific role in the signaling involved in this process was unknown. It was also unknown on what time scale Shp2 regulates the migration of cells and what specific morphodynamic changes it induces at different time points of its activation. Further, it was unclear whether acute and prolonged activation of Shp2 will cause different effects. Using RapR-Shp2, it was found that acute activation of Shp2 induces transient cell spreading, an increase in protrusions, cell polarization, and migration. Specific pathways downstream of Shp2 that regulate distinct morphodynamic changes were also identified12. This protocol provides details for the design and characterization of RapR-Shp2, which can be used to guide the development and application of other RapR phosphatases.

<meta charset="utf8"></head><body>저자 스포트라이트: 티로신 인산가수분해효소의 활성을 조정하기 위한 도구 개발

Rapamycin-regulated Tyrosine Phosphatase의 개발 및 응용

Our work focuses on the development of tools that regulate the activity of tyrosine phosphatases. We've applied the rapamycin-regulated system to several phosphatases and shown that it imparts specific and temporal regulation of our protein of interest. We've aimed to determine the morphological changes induced by the activation of SHIP-2 phosphatase.The RapR tool has allowed us to determine transient effects of phosphatase activation in cells, and we've determined complex specific downstream effects of a phosphatase involved in cell spreading and migration. Additionally, we've determined domain-driven behaviors of the same phosphatase. This has contributed to our understanding of phosphatase mechanisms in regulating cell morphodynamics and downstream signaling.Our RapR tool offers the ability to specifically and temporally regulate a phosphatase of interest. Once inserted into the phosphatase, the iFKBP domain acts as an allosteric switch that, once bound to rapamycin, restores activity to the phosphatase, acting as an on-switch. We can also use the rapamycin-regulated system to reconstruct signaling complexes.Our findings using the RapR tool have opened the door to determining the effects of SHIP-2's interactome on its ability to regulate cell activity. We found that SHIP-2s in SH2 domain binding is crucial in cell retraction. Using the RapR tool, we can further investigate what binding partners may be responsible for this regulation.

Tyrosine phosphatases are an important family of enzymes that regulate critical physiological functions. They are often dysregulated in human diseases, ...

development-application-rapamycin-regulated-tyrosine

Research

JoVE Journal

Biology

Development and Application of Rapamycin-regulated Tyrosine Phosphatases

142 조회수.  University of Illinois at Chicago. 우리의 연구는 티로신 인산가수분해효소의 활성을 조절하는 도구 개발에 중점을 두고 있습니다. 우리는 라파마이신 조절 시스템을 여러 인산가수분해효소에 적용하였고, 이것이 우리의 관심 단백질에 대한 특이적이고 시간적인 조절을 부여한다는 것을 보여주었습니다. 우리는 SHIP-2 인산가수분해효소의 활성화에 의해 유도되는 형태학적 변화를 결...

비디오: 저자 스포트라이트: 티로신 인산가수분해효소의 활성을 조정하기 위한 도구 개발

Tyrosine phosphatases are an important family of enzymes that regulate critical physiological functions. They are often dysregulated in human diseases, making them key targets of biological studies. Tools that enable the regulation of phosphatase activity are instrumental in the dissection of their function. Traditional approaches, such as overexpression of constitutively active or dominant negative mutants, or downregulation using siRNA, lack temporal control. Phosphatase inhibitors often have poor specificity, and they only allow researchers to determine what processes are affected by the inhibition of the phosphatase. 
We developed a chemogenetic approach, the Rapamycin-regulated (RapR) system, which allows for allosteric regulation of a phosphatase catalytic domain that enables tight temporal control of phosphatase activation. The RapR system consists of an iFKBP domain inserted into an allosteric site in the phosphatase. The intrinsic structural dynamics of the RapR domain disrupt the catalytic domain, leading to the inactivation of the enzyme. The addition of rapamycin mediates the formation of a complex between iFKBP and a co-expressed FRB protein, which stabilizes iFKBP and restores activity to the phosphatase's catalytic domain. 
This system provides high specificity and tight temporal control of phosphatase activation in living cells. The unique capabilities of this system enable the identification of transient events and interrogation of individual signaling pathways downstream of a phosphatase. This protocol describes guidelines for the development of a RapR-phosphatase, its biochemical characterization, and the analysis of its effects on downstream signaling and regulation of cell morphodynamics. It also provides a detailed description of a protein engineering strategy, in vitro assays analyzing phosphatase activity, and live cell imaging experiments identifying changes in cell morphology.

This protocol describes the design, creation, and application of rapamycin-regulated phosphatases. This method provides high specificity and tight temporal control of phosphatase activation in living cells.

연구

생물학

Preparing Cells Expressing Rapamycin-Regulated Phosphatase for Immunoprecipitation to Assess Temporal Control of Tyrosine Phosphatases

To begin, obtain HEK 293T cells and plate 800, 000 cells per well in a 6-well tissue culture plate in supplemented DMEM media. Incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide to achieve 60 to 80%confluency. Next, mix one microgram of each plasmid DNA with 200 microliters of serum-free DMEM and six microliters of transfection reagent.After 15 minutes of incubation, dispense 100 microliters of transfection mixture per well of cells. Incubate overnight in a 37 degrees Celsius and 5%carbon dioxide incubator. On day three, add an appropriate amount of either rapamycin or ethanol separately to the cells and incubate them for one hour in a 37 degrees Celsius and 5%carbon dioxide incubator.Then place the plate on ice and gently wash the cells with three milliliters of cold PBS. After removing the PBS add 300 microliters of lysis buffer containing either rapamycin or ethanol, and scrape the cells with a cell scraper. Transfer the sample to a 1.5 milliliter tube and microcentrifuge it for 10 minutes at 1, 000 g at four degrees Celsius.To check for expression levels, aliquot 20 microliters of the supernatant into a new 1.5 milliliter tube. Incubate the sample with 20 microliters of 2x Laemmli buffer containing 5%beta mercaptoethanol at 95 to 100 degrees Celsius for five minutes.

티로신 포스파타제의 시간적 제어를 평가하기 위해 면역침전을 위해 라파마이신 조절 인산가수분해효소를 발현하는 세포 준비

Immunoprecipitation of RapR-Shp2 to Study Chemogenetic Control of Tyrosine Phosphatase

To begin, culture and prepare the HEK 293 T-cells expressing RapR-Shp2 for immunoprecipitation. For preparing the Sepharose, add resuspended Protein G-Sepharose into a 1.5 milliliter tube. After adding the lysis buffer, micro-centrifuge the tube for one minute at 2000 G and carefully remove the lysis buffer.Then resuspend the Sepharose in 380 microliters of lysis buffer by inverting the tube a few times. Incubate the Sepharose with BSA and the antibody on a rotator at four degrees Celsius for 1.5 hours. After washing the Sepharose as demonstrated earlier, use a cut tip to carefully resuspend it in the lysis buffer.Add the supernatant from the prepared HEK 293 T-cells expressing RapR-Shp2 to 50 microliters of the suspended Sepharose in a new 1.5 milliliter tube, and incubate the tube on a rotator at four degrees Celsius for 1.5 to two hours.

티로신 인산가수분해효소의 화학유전학적 제어를 연구하기 위한 RapR-SHP2의 면역침전

Phosphatase Activity Assay to Characterize Allosteric Regulation of Phosphatases with the Rapamycin-Regulated RapR System

To begin, obtain the HEK293T cells expressing RapR-Shp2 and perform immunoprecipitation with Protein G-Sepharose. After immunoprecipitation, wash the sepharose two times each with 0.5 milliliters of lysis buffer, followed by 0.5 milliliters of wash buffer. Remove as much buffer as possible after the final spin.Then add 40 microliters of the phospho-paxillin in imidazole buffer mixture to each sample with or without one micromolar rapamycin. Flick the tube gently and immediately place it in the heating shaker for 40 minutes at 32 degrees Celsius. Set the heating shaker to 1000 revolutions per minute to ensure full agitation of the sample.To stop the reaction, add 40 microliters of 2X Laemmli buffer to each sample and incubate them at 95 to 100 degrees Celsius for five minutes. After the samples cool down, load 15 microliters of each sample onto a four to 15%gradient SDS polyacrylamide gel. Run a standard Western blot protocol using PVDF membranes.Finally, blot using anti-FLAG antibody to detect the RapR-Shp2 construct and anti-phospho-paxillin to detect phosphorylated paxillin in the samples. Active RapR-Shp2 showed no phospho-paxillin, similar to the constitutively active Shp2, indicating retained full activity. Inactive RapR-Shp2 and dominant negative Shp2 showed similar phospho-paxillin levels, indicating no activity when inactive.