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

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

DOI:
10.3791/62608-v

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11:38 min

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September 14, 2021

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, ,
1Institute for Chemistry and Biochemistry,Freie Universität Berlin, 2International Max-Planck Research School for Biology and Computation

Capítulos

  • 00:05Introduction
  • 00:40Cell Culture and Fluid Shear Stress Exposure
  • 02:55Fixation
  • 04:58Blocking and Incubation with Primary Antibody
  • 06:28Incubation with PLA Probe and Ligation
  • 07:34Amplification and Mounting
  • 09:14Results: Effect of Shear Stress on the SMAD Protein Interactions
  • 11:02Conclusion

Summary

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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.

Tags

Keywords: TGF-β/BMP/SMAD SignalingProximity Ligation AssayFluid Shear StressMicrofluidic Flow ChannelsHUVEC CellsTranscription Factor ComplexesVascular Biology

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