Figure 3.
Platelets express truncated TrkB receptors. (A) Immunoblotting of TrkB from human platelet lysates (100 µg for Sino Biological clone 7H6E7B3 and 25 µg for Abnova clone 3D12) obtained from 4 different healthy volunteers. TrkB-Fc fusion protein (5 ng; expected molecular weight, 120 kDa) and human cortex whole cell lysate (5 µg; expected molecular weight of truncated TrkB and the full-length TrkB receptor, 95- and 140-kDa, respectively) were used as positive controls. β-Actin was used a loading control. Two different antibodies (clone 7H6E7B3 and clone 3D12) targeting the extracellular domain of TrkB were used. Blots are representative of ≥3 independent experiments. (B) Confocal fluorescence microscopy of platelets expressing TrkB. Washed platelets, PBMCs, U251-MG cells, and HEPG2 cells were labeled with anti-TrkB primary antibodies and Alexa Fluor 488–conjugated secondary antibodies. Nuclei of PBMCs, U251-MG cells, and HEPG2 cells were stained with 4′,6-diamidino-2-phenylindole, and IgG2B/IgG1 was used as an isotype control. Images were visualized at room temperature with Zeiss LSM510 using a 100× objective lens for platelets and PBMCs, a 63× objective lens for HEPG2 cells, and a 20× objective lens for U251-MG cells as well as 3× magnification. Scale bar represents 5 µm for PBMCs and platelets, 10 µm for HEPG2 cells, and 200 µm for U251-MG cells. Images are representative of 3 independent experiments. (C) Flow cytometry of surface and intracellular TrkB on washed human platelets; platelets expressed TrkB on both their surface (TrkB+: 27% ± 11%, n = 8) and their intracellular compartment (TrkB+: 82% ± 9%, n = 6). PBMCs were used as positive controls for TrkB labeling on both the surface (TrkB+: 45% ± 15%, n = 4) and the intracellular compartment (TrkB+: 88% ± 6%, n = 4). U251-MG cells were used as positive controls for TrkB labeling on both the surface (TrkB+: 30% ± 2%, n = 3) and the intracellular compartment (TrkB+: 94% ± 2%, n = 3). IgG1/2B was used as isotype control. HEPG2 cells were used as TrkB-low controls for TrkB labeling on both the surface (TrkB+: 6% ± 0.12%, n = 2) and the intracellular compartment (TrkB+: 17% ± 1%, n = 2). FITC, fluorescein isothiocyanate.

Platelets express truncated TrkB receptors. (A) Immunoblotting of TrkB from human platelet lysates (100 µg for Sino Biological clone 7H6E7B3 and 25 µg for Abnova clone 3D12) obtained from 4 different healthy volunteers. TrkB-Fc fusion protein (5 ng; expected molecular weight, 120 kDa) and human cortex whole cell lysate (5 µg; expected molecular weight of truncated TrkB and the full-length TrkB receptor, 95- and 140-kDa, respectively) were used as positive controls. β-Actin was used a loading control. Two different antibodies (clone 7H6E7B3 and clone 3D12) targeting the extracellular domain of TrkB were used. Blots are representative of ≥3 independent experiments. (B) Confocal fluorescence microscopy of platelets expressing TrkB. Washed platelets, PBMCs, U251-MG cells, and HEPG2 cells were labeled with anti-TrkB primary antibodies and Alexa Fluor 488–conjugated secondary antibodies. Nuclei of PBMCs, U251-MG cells, and HEPG2 cells were stained with 4′,6-diamidino-2-phenylindole, and IgG2B/IgG1 was used as an isotype control. Images were visualized at room temperature with Zeiss LSM510 using a 100× objective lens for platelets and PBMCs, a 63× objective lens for HEPG2 cells, and a 20× objective lens for U251-MG cells as well as 3× magnification. Scale bar represents 5 µm for PBMCs and platelets, 10 µm for HEPG2 cells, and 200 µm for U251-MG cells. Images are representative of 3 independent experiments. (C) Flow cytometry of surface and intracellular TrkB on washed human platelets; platelets expressed TrkB on both their surface (TrkB+: 27% ± 11%, n = 8) and their intracellular compartment (TrkB+: 82% ± 9%, n = 6). PBMCs were used as positive controls for TrkB labeling on both the surface (TrkB+: 45% ± 15%, n = 4) and the intracellular compartment (TrkB+: 88% ± 6%, n = 4). U251-MG cells were used as positive controls for TrkB labeling on both the surface (TrkB+: 30% ± 2%, n = 3) and the intracellular compartment (TrkB+: 94% ± 2%, n = 3). IgG1/2B was used as isotype control. HEPG2 cells were used as TrkB-low controls for TrkB labeling on both the surface (TrkB+: 6% ± 0.12%, n = 2) and the intracellular compartment (TrkB+: 17% ± 1%, n = 2). FITC, fluorescein isothiocyanate.

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