We thank Dr. Maria Reichenbach and Dr. Christian Hiepen for their support on the flow-set up system and Eleanor Fox and Yunyun Xiao for critically reading the manuscript. P-L.M. was funded by the international Max Planck Research School IMPRS-Biology and Computation (IMPRS-BAC). PK received funding by the DFG-SFB1444. Figure 1 was created using BioRender.

1. Cell culture and fluid shear stress exposure
NOTE: Human umbilical vein ECs (HUVECs) were used as an example to study SS induced interaction of SMADs. The protocol described below can be applied to every SS responsive cell type.
<ol>
	<li>Coat 6-channel slide with 0.1% porcine skin gelatin in PBS for 30 min at 37 &#176;C.</li>
	<li>Seed HUVECs in pre-coated 6-channel slides at a density of 2.5 x 106 cells per mL in 30 &#181;L of M199 full medium. 
	NOTE: For further information on how to seed cells in the flow slide, see reference24.</li>
	<li>Let cells adhere for 1 h at 37 &#176;C in a humidified incubator.</li>
	<li>Add 60 &#181;L of pre-warmed M199 full medium to each of the reservoirs.</li>
	<li>Culture for 2 days, with a gentle medium exchange once a day, at 37 &#176;C in a humidified incubator.
	<ol>
		<li>Aspirate the reservoirs completely, add 120 &#181;L of pre-warmed M199 full medium in one of the reservoirs, and aspirate from the other side.</li>
		<li>Add 60 &#181;L of pre-warmed M199 full medium to both reservoirs.</li>
	</ol>
	</li>
	<li>Assemble and start the flow set-up as detailed in the reference25.
	<ol>
		<li>Mount tubing on fluidic units. Here, silicone tubing with a diameter of 0.8 mm and 1.6 mm are used to apply shear stress of 1 dyn/cm2 and 30 dyn/cm2, respectively. 
		NOTE: The material and tubing length should remain constant, as changes could influence the resulting shear stress. In general, other combinations of pump systems and tubing can be used, as long as the resulting shear stress is known, and the pump creates a steady laminar flow.</li>
		<li>Fill the reservoirs with an appropriate amount of pre-warmed M199 full medium (minimum 10 mL).</li>
		<li>Connect fluidic units with the tubing to the pump system and perform a pre-run without cells to equilibrate the medium and to remove any remaining air25.</li>
		<li>Serially connect the single channels on the 6-channel slide to one another by using serial connection tubing. The first and the last channel on the slide will be connected to the tubing assembled in 1.6.1 (see Figure 1A for a scheme). Be careful not to introduce any air into the system as this could severely harm the cells. Further information on the serial connection can be found in reference26.</li>
		<li>For the exposure of cells to high levels of shear stress (&#62;20 dyn/cm2), use a ramp phase, i.e., increase the shear stress stepwise with adaptation phases. Steps can be set in increments of 5 dyn/cm2 per 30 min.</li>
	</ol>
	</li>
</ol>
2. Fixation
<ol>
	<li>Detach slides from the pumps after the fluid SS exposure. Use clamps on the tubing when detaching, to avoid the medium spill in the incubator.</li>
	<li>Immediately transfer flow slides on ice, while the remaining tubing is detached sequentially. When removing the tubing from reservoirs, the reservoir on the other side should be kept closed with a finger to avoid trapping air bubbles in the channel. This might interfere with fixation steps.</li>
	<li>Keeping the cells on ice, aspirate the medium carefully from the reservoirs but not from the channel where the cells reside. Subsequently, wash samples with cold sterile PBS (4 &#176;C) with three times the channel volume (90 &#181;L). Add PBS in one reservoir and aspirate carefully from the other reservoir. Repeat this step in each of the 6 channels per slide. 
	NOTE: For all washing and incubation steps the respective solution is added in one of the reservoirs which leads to an exchange of solutions in the channel. To allow for complete substitution of solutions in the channel, the excess solution is then aspirated from the other reservoir. Solution on the top of the cells in the channel is not removed. Cells should not dry at any time. Therefore, it is important to wash carefully without any air bubble insertion into the slides.</li>
	<li>Fix the cells by adding 90 &#181;L of buffered 4% PFA solution in the same reservoir where the PBS was added beforehand and similarly aspirate the liquid from the other reservoir. Repeat this step in each channel in each slide. After the addition of PFA solution, transfer the samples from ice to room temperature (RT) and incubate for 20 min. 
	CAUTION: PFA is toxic and should be handled carefully. Use gloves and work under a fume hood.</li>
	<li>Wash cells 3x with PBS (RT) by adding it in one reservoir and aspirating carefully from the other reservoir. Empty just the reservoirs, ensuring not to dry out the channel. Repeat this step for each of the 6 channels per slide.</li>
	<li>Quench the PFA-fixation by adding 90 &#181;L of ambient 50 mM ammonium chloride in PBS in one of the reservoirs. Aspirate excess solution from the other reservoir. Repeat for each channel in the slide. Incubate the samples for 10 min at RT.</li>
	<li>Wash as described in step 2.5. 
	&#8203;NOTE: At this point, the samples may be stored at 4 &#176;C overnight, or the protocol can be immediately continued with blocking and primary antibody incubation (see step 3).</li>
</ol>
3. Blocking and primary antibody incubation
<ol>
	<li>To permeabilize the cells, add 90 &#181;L of 0.3% Triton-X-100 in PBS in an emptied reservoir, and aspirate from the other reservoir for each channel. Incubate for 10 min at RT.</li>
	<li>Wash as described in step 2.5.</li>
	<li>Add 90 &#181;L of sterile PLA blocking solution in one reservoir of a channel and aspirate from the other side. Repeat this step for each channel. Block for 1 h at 37 &#176;C in a humidified chamber.
	<ol>
		<li>To make a humidified chamber, use a 10 cm dish with wet tissue sealed with wax film and place the dish in the incubator. Alternatively, other humidity chamber formats can be used that supply a humid atmosphere. 
		NOTE: Alternatively, self-made blocking solution can be used (e.g., 3% (w/v) BSA in PBS, sterile filtered).</li>
	</ol>
	</li>
	<li>Prepare primary antibodies (1:100) in PLA antibody diluent. Prepare 30 &#181;L of the solution per channel. Add both primary antibodies simultaneously and vortex. 
	NOTE: Alternatively, self-made antibody diluent can be used (e.g., 1% (w/v) BSA in PBS). Antibodies used here are combinations of SMAD1-SMAD2/3, SMAD2/3-SMAD4 and phospho-SMAD1/5-SMAD4. Detailed information can be found in the Table of Materials.</li>
	<li>Before the application of primary antibodies, aspirate the blocking solution from the reservoirs and, also, carefully from the channel. Pipette 30 &#181;L of the primary antibody solution immediately into the empty channel by tilting the channel while adding the solution. 
	NOTE: Perform the removal of the blocking solution and addition of the antibody solution channel-by-channel to ensure cells do not dry out in between.</li>
	<li>Incubate samples with the primary antibodies overnight in humidified chambers at 4 &#176;C. 
	&#8203;NOTE: The incubation can also be performed for 1 h at room temperature, if interested in continuing with the following steps on the same day.</li>
</ol>
4. PLA probe incubation
NOTE: For all steps in section 4.1-7.3, the washing buffers A and B are stored at 4 &#176;C and need to be warmed to RT prior to the use.
<ol>
	<li>Dilute PLA-probes (+)-mouse and (-)-rabbit to 1:5 in PLA antibody diluent (or 1% BSA) solution. Prepare 30 &#181;L per channel.</li>
	<li>Wash samples 2x for 5 min using 90 &#181;L of the wash buffer A at RT by adding it in one of the reservoirs and aspirating carefully from the other reservoir. Repeat this step for each of the 6 channels per slide.</li>
	<li>Aspirate the wash buffer A carefully and add 30 &#181;L of PLA probe solution (prepared in step 4.1), similar to the addition of primary antibodies in step 3.5.</li>
	<li>Incubate samples for 1 h at 37 &#176;C in a humidified chamber.</li>
</ol>
5. Ligation
<ol>
	<li>Wash samples 2x for 5 min using 90 &#181;L of the wash buffer A at RT, as described in 4.2.</li>
	<li>Prepare a 1:5 dilution of the ligation buffer in deionized water. Use this buffer to dilute the ligase enzyme to 1:40 (on ice). Use 30 &#181;L per channel.</li>
	<li>Aspirate the wash buffer A completely and add the ligation solution as described in 3.5.</li>
	<li>Incubate samples for 30 min at 37 &#176;C in a humidified chamber.</li>
</ol>
6. Amplification
<ol>
	<li>Wash samples 2x for 2 min using 90 &#181;L of wash buffer A at RT, as described in 4.2.</li>
	<li>Prepare the amplification buffer by diluting it 1:5 in deionized water and use it to dilute the polymerase enzyme to 1:80 (on ice). Protect from light. Prepare 30 &#181;L per channel.</li>
	<li>Aspirate the wash buffer A completely and immediately add the prepared amplification solution into the empty channel, as described in 3.5. Incubate samples for 100 min at 37 &#176;C in a humidified chamber.</li>
</ol>
7. Mounting
<ol>
	<li>Wash samples 2x for 10 min using 90 &#181;L of Wash Buffer B at RT as described in 4.2. Add DAPI (1:500) from 1 mg/mL stock solution (in deionized water) in the first wash to stain nuclei. Do not dry the channel.</li>
	<li>Dilute the wash buffer B in deionized water (1:10) and wash 1x with 90 &#181;L of 0.1x buffer B solution as described in 4.2.</li>
	<li>Aspirate the wash buffer B completely and immediately add 2-3 drops of the liquid mounting medium into one reservoir. Distribute it in the channel by tilting the slide. Store samples at 4 &#176;C in a humidified environment until imaging.</li>
</ol>
8. Image acquisition
<ol>
	<li>Acquire images using a fluorescence microscope. Ensure that the respective filters fitting the fluorescent PLA probes are available. 
	NOTE: It is beneficial to make use of a confocal microscope, if possible, as obtained PLA spots are more defined. This also supports further image processing and data analysis.</li>
</ol>
9. Image analysis and quantification using ImageJ/FIJI
<ol>
	<li>Process exported images (.tiff) with an image processing program, such as ImageJ27. 
	NOTE: All scripts used within this study and that are necessary for the automatic counting of cellular, nuclear, and all PLA events (per cell) can be found in a GitHub repository: https://github.com/Habacef/Proximity-Ligation-Assay-analysis. Perform statistical analysis using any suitable program or tool.</li>
</ol>

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The authors declare no conflict of interest.

The PLA based protocol described here offers an efficient way to determine close proximity of two proteins (e.g., their direct interaction) in ECs exposed to shear stress with quantitative and spatial resolution. By using flow slides with multiple parallel channels, several different protein interactions can be examined at the same time in cells under identical mechanical conditions. In contrast, custom-build flow chamber systems often make use of a single channel that is built around a glass coverslip, which would allow...

Proteins of the transforming growth factors beta (TGF&#946;) superfamily are pleiotropic cytokines with a variety of members, including TGF&#946;s, bone morphogenetic proteins (BMPs), and Activins1,2. Ligand binding induces the formation of receptor oligomers leading to the phosphorylation and, thereby, activation of cytosolic regulatory SMAD (R-SMAD). Depending on the sub-family of ligands, different R-SMADs are activated1,2. While TGF&#946;s and Activins mainly induce phosphorylation of SMAD2/3, BMPs induce SMAD1/5/8 phosphorylation. However, there are accumulating evidences that BMPs and TGF&#946;s/Activins also activate R-SMADs of the respective other sub-family, in a process termed as &#39;lateral signaling&#39;3,4,5,6,7,8 and that there are mixed SMAD complexes consisting of both, SMAD1/5 and SMAD2/3, members3,9. Two activated R-SMADs subsequently form trimeric complexes with the common mediator SMAD4. These transcription factor complexes are then able to translocate into the nucleus and regulate the transcription of target genes. SMADs can interact with a variety of different transcriptional co-activators and co-repressors, leading to the diversification of the possibilities to regulate target genes10. Deregulation of SMAD signaling has severe implications in a variety of diseases. In line with this, unbalanced TGF&#946;/BMP signaling may lead to severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, or atherosclerosis3,11,12,13,14.
Endothelial cells (ECs) form the innermost layer of blood vessels and are, therefore, exposed to shear stress (SS), a frictional force exerted by the viscous flow of the blood. Interestingly, ECs residing at the parts of the vasculature, which are exposed to high levels of uniform, laminar SS, are kept in a homeostatic and quiescent state. In contrast, ECs that experience low, non-uniform SS, e.g., at bifurcations or the lesser curvature of the aortic arch, are proliferative and activate inflammatory pathways15. In turn, sites of dysfunctional ECs are prone to develop atherosclerosis. Interestingly, ECs in these atheroprone areas display aberrantly high levels of activated SMAD2/3 and SMAD1/516,17,18. In this context, enhanced TGF&#946;/BMP signaling was found to be an early event in the development of atherosclerotic lesions19 and interference with BMP signaling was found to markedly reduce vascular inflammation, atheroma formation, and associated calcification20.
Proximity Ligation Assay (PLA) is a biochemical technique to study protein-protein interactions in situ21,22. It relies on the specificity of antibodies of different species that can bind target proteins of interest, allowing highly specific detection of endogenous protein interactions at a single-cell level. Here, primary antibodies have to bind to their target epitope at a distance of less than 40 nm to allow for the detection23. Therefore, PLA is greatly beneficial over traditional co-immunoprecipitation approaches, where several million cells are needed to detect endogenous protein interactions. In PLA, species-specific secondary antibodies, covalently linked to DNA fragments (termed Plus and Minus probes), bind the primary antibodies and if the proteins of interest interact, Plus and Minus probes come in close proximity. The DNA gets ligated in the following step and the rolling circle amplification of the circular DNA is made possible. During amplification, fluorescently labeled complementary oligonucleotides bind to the synthesized DNA, allowing these protein interactions to be visualized by conventional fluorescence microscopy.
The protocol described here enables scientists to quantitatively compare the number of active SMAD transcription complexes at atheroprotective and atheroprone SS conditions in vitro using PLA. SS is generated via a programmable pneumatic pump system that is able to generate laminar unidirectional flow of defined levels and allows stepwise increases of flow rates. This method allows for the detection of interactions between SMAD1/5 or SMAD2/3 with SMAD4, as well as mixed-R-SMAD complexes. It can easily be expanded to analyze interactions of SMADs with transcriptional co-regulators or to transcription factor complexes other than SMADs. Figure 1 shows the major steps of the protocol presented below.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62608/62608fig01.jpg" /> 
Figure 1: Schematic representation of the protocol described. (A)&#160;Cells seeded in 6-channel slides are exposed to shear stress with a pneumatic pump system. (B)&#160;Fixed cells are used for PLA experiment or for control conditions. (C)&#160;Images of PLA experiments are acquired with a fluorescence microscope and are analyzed using ImageJ analysis software. <a href="https://www.jove.com/files/ftp_upload/62608/62608fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Slide VI 0.4</td>
			<td>ibidi</td>
			<td>80606</td>
			<td>6-channel slide</td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>Carl Roth</td>
			<td>K298.1</td>
			<td>Quenching</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Carl Roth</td>
			<td>8076.4</td>
			<td>Blocking</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich/ Merck</td>
			<td>D9542</td>
			<td>Stain DNA/Nuclei</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>PAN Biotech</td>
			<td>P04-53500</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Duolink In Situ Detection Reagents Green</td>
			<td>Sigma Aldrich/ Merck</td>
			<td>DUO92014</td>
			<td>PLA kit containing Ligase, ligation buffer, polymerase and amplification buffer (with green labeled oligonucleotides)</td>
		</tr>
		<tr>
			<td>Duolink In Situ PLA Probe Anti-Mouse MINUS</td>
			<td>Sigma Aldrich/ Merck</td>
			<td>DUO92004</td>
			<td>MINUS probe</td>
		</tr>
		<tr>
			<td>Duolink In Situ PLA Probe Anti-Rabbit PLUS</td>
			<td>Sigma Aldrich/ Merck</td>
			<td>DUO92002</td>
			<td>PLUS probe</td>
		</tr>
		<tr>
			<td>Duolink In Situ Wash Buffers, Fluorescence</td>
			<td>Sigma Aldrich/ Merck</td>
			<td>DUO82049</td>
			<td>PLA wash buffers A and B</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Supplement</td>
			<td>Corning</td>
			<td></td>
			<td>supplement for medium (ECGS)</td>
		</tr>
		<tr>
			<td>Fetal calf Serum</td>
			<td></td>
			<td></td>
			<td>supplement for medium</td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td>Image Analysis software</td>
		</tr>
		<tr>
			<td>Formaldehyde solution 4% buffered</td>
			<td>KLINIPATH/VWR</td>
			<td>VWRK4186.BO1</td>
			<td>PFA</td>
		</tr>
		<tr>
			<td>Full medium</td>
			<td></td>
			<td></td>
			<td>M199 basal medium +20 % FCS +1 % P/S + 2 nM L-Glu +&#160; 25 &#181;g/mL Hep +&#160;&#160; 50 &#181;g/mL ECGS</td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin, Type A</td>
			<td>Sigma Aldrich</td>
			<td>G2500</td>
			<td>Use 0.1% in PBS for coating of flow channels</td>
		</tr>
		<tr>
			<td>GraphPad Prism v.7</td>
			<td>GarphPad</td>
			<td></td>
			<td>Statistical Program used for the Plots and statistical calculations</td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma Aldrich</td>
			<td>H4784-250MG</td>
			<td>supplement for medium (Hep)</td>
		</tr>
		<tr>
			<td>HUVECs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ibidi Mounting Medium</td>
			<td>ibidi</td>
			<td>50001</td>
			<td>Liquid mounting medium</td>
		</tr>
		<tr>
			<td>ibidi Pump System</td>
			<td>ibidi</td>
			<td>10902</td>
			<td>pneumatic pump</td>
		</tr>
		<tr>
			<td>Leica TCS SP8</td>
			<td>Leica</td>
			<td></td>
			<td>confocal microscope</td>
		</tr>
		<tr>
			<td>L-Glutamin 200mM</td>
			<td>PAN Biotech</td>
			<td>P04-80100</td>
			<td>supplement for medium (L-Glu)</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma Aldrich</td>
			<td>M2154</td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>mouse anti- SMAD1 Antibody</td>
			<td>Abcam</td>
			<td>ab53745</td>
			<td>Suited for PLA</td>
		</tr>
		<tr>
			<td>mouse anti- SMAD2/3 Antibody</td>
			<td>BD Bioscience</td>
			<td>610843</td>
			<td>Not suited for PLA in combination with CST 9515</td>
		</tr>
		<tr>
			<td>mousee anti- SMAD4 Antibody</td>
			<td>Sanata Cruz Biotechnology</td>
			<td>sc-7966</td>
			<td>Suited for PLA</td>
		</tr>
		<tr>
			<td>Penicillin 10.000U/ml /Streptomycin 10mg/ml</td>
			<td>PAN Biotech</td>
			<td>P06-07100</td>
			<td>supplement for medium (P/S)</td>
		</tr>
		<tr>
			<td>Perfusion Set WHITE</td>
			<td>ibidi</td>
			<td>10963</td>
			<td>Tubings used for 1 dyn/cm2</td>
		</tr>
		<tr>
			<td>Perfusion Set YELLOW and GREEN</td>
			<td>ibidi</td>
			<td>10964</td>
			<td>Tubings used for 30 dyn/cm2</td>
		</tr>
		<tr>
			<td>rabbit anti- phospho SMAD1/5 Antibody</td>
			<td>Cell Signaling Technologies</td>
			<td>9516</td>
			<td>Suited for PLA</td>
		</tr>
		<tr>
			<td>rabbit anti- SMAD2/3 XP Antibody</td>
			<td>Cell Signaling Technologies</td>
			<td>8685</td>
			<td>Suited for PLA</td>
		</tr>
		<tr>
			<td>rabbit anti- SMAD4 Antibody</td>
			<td>Cell Signaling Technologies</td>
			<td>9515</td>
			<td>Not suited for PLA in combination with BD 610843</td>
		</tr>
		<tr>
			<td>Serial Connector for &#181;-Slides</td>
			<td>ibidi</td>
			<td>10830</td>
			<td>serial connection tubes</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>6683.1</td>
			<td>Permeabilization</td>
		</tr>
	</tbody>
</table>

visualization and quantification of tgf&#946;/bmp/smad signaling under different fluid shear stress conditions using proximity-ligation-assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

We have previously used PLA to detect interactions of different SMAD proteins3 and analyzed shear stress induced changes in SMAD phosphorylation28.
Here, both methods were combined with the protocol described above. HUVECs were subjected to shear stress of 1 dyn/cm2 and 30 dyn/cm2 and analyzed for interactions of SMAD transcription factors. We show that, when compared to the high shear stress (30 dyn/cm2), the low...

Watch this Scientific Journal Video about Visualization and Quantification of TGFÃŽÂ²/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay at JoVE.com

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...

visualization-quantification-tgfbmpsmad-signaling-under-different

Research

JoVE Journal

Biochemistry

3.2K Views.  Freie Universität Berlin. Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Video: Visualization and Quantification of TGFβ/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.