Journal: The Journal of Biological Chemistry
Article Title: Evidence for shear-mediated Ca 2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line
doi: 10.1074/jbc.M116.766196
Figure Lengend Snippet: The Piezo1 agonist Yoda1 induced increases in [Ca 2+ ] i in platelets and Meg-01 cells. A–C , [Ca 2+ ] i responses to Yoda1 (25 μ m ) assessed in stirred Fura-2-loaded washed suspensions of platelets ( top panels ) and Meg-01 cells ( bottom panels ). A and B show representative recordings, and C shows the average peak [Ca 2+ ] i increases ( n = 4) for Yoda1 in the presence of extracellular Ca 2+ compared with its vehicle control (DMSO) and following removal of external Ca 2+ (EGTA). D , representative intracellular Ca 2+ recording (Fluo-3 F / F 0 fluorescence) from a single platelet attached to a PECAM-1-coated glass coverslip in the presence of Yoda1 and exposed to two cycles of no flow ( white regions ) and arterial shear ( gray regions ). See C , ( panel i ) for the control trace. E , average Ca 2+ increases above baseline ( F / F 0 ·4 min) in the presence and absence of Yoda1 under conditions of no flow and normal arterial shear ( n = 20, 35, 20, and 35 cells in HBSS no flow, Yoda1 no flow, HBSS normal arterial, and Yoda1 normal arterial conditions, respectively). **, p < 0.01; †, p < 0.01 compared with no flow, in the presence of HBSS; ‡, p < 0.001 compared with no flow in presence of Yoda1. Apyrase was omitted from the extracellular buffer to avoid P2X1 receptor responses (see ).
Article Snippet: The antibody concentrations used were: FAM38A (Piezo1) rabbit anti-human polyclonal antibody (15939-1-AP; ProteinTech, Manchester, UK), 1:2000; P2X1 rabbit anti-human polyclonal antibody (APR-001; Alomone Labs, Israel), 1:1000; and FAM38B (Piezo2) rabbit anti-human polyclonal antibody (G-20 Santa Cruz Biotechnology, Heidelberg, Germany), 1:200.
Techniques: Fluorescence