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. 2021 Feb 19;40(9):e106113. doi: 10.15252/embj.2020106113

Figure EV3. Leukocyte‐induced tension across VE‐cadherin is independent of SHP2, and tension is not required for SHP2 dissociation from PECAM‐1.

Figure EV3

  1. Quantification of FRET efficiency (percentage) at junctions of HUVEC treated with siRNAs for SHP2 and VE‐cadherin prior to transducing VE‐cadherin‐FL. Cells were exposed either to flow alone or to flow in the presence of PMNs and then fixed with 4% PFA followed by FLIM measurements at sites of PMN transmigration (PMN) or at junctions without PMNs (Flow).
  2. Representative immunoblot for SHP2 and actin expression of HUVEC used for the experiments in (A).
  3. HUVEC were grown on collagen‐coated polyacrylamide gels of varying physiologic stiffness of 0.2 kPa (left) and 20 kPa (right) and stimulated with TNF‐α for 17 h prior to adding HL60‐derived neutrophils for 20 min. PECAM‐1 immunoprecipitates and cell lysates were immunoblotted for the indicated antigens. Note that leukocytes triggered SHP2 dissociation from PECAM‐1 independent of substrate stiffness.

Data information: Graph in (A) represents n = 21 and 20 measurements in a total of four independent experiments. Bars and error bars indicate mean ± SEM. Statistical significance was tested with unpaired t‐test, **P < 0.01. Blots are representative for 3 experiments (C).