Fig 1: Vascular and cavernosal reactivity to Piezo1 activation induced by Yoda1. Relaxation curves to Yoda1 in the pudendal artery and corpus cavernosum incubated with Dooku (A, B); (n = 5), and GsMTx4 (C, D); (n = 5). The magnitude of the relaxation induced by Yoda1 was calculated relative to the maximal changes from the contraction produced by PE, which was taken as 100%. Results are presented as mean ± SEM. *p < 0.05 compared to vehicle.
Fig 2: Representative blot (A) and protein expression (B) of Piezo1 in the pudendal artery (left panel, n = 4) and corpus cavernosum (right panel, n = 4), respectively.
Fig 3: Single-channel recordings of Piezo1 activity in representative whole-cell experiments on K562 cells. (A) Extracellular application of Yoda1 resulted in the activation of Piezo1 in the whole-cell membrane. The replacement of all permeable cations with non-permeable NMDG+ resulted in the full abolition of Yoda1-induced ion currents. The amplitude histogram calculated for the particular current trace is presented below. Single-channel Piezo1 amplitudes could be clearly resolved between the centers of the peaks of the histograms. (B) The I-V relationship of Piezo1 channels. The mean unitary conductance of the channels is 19.3 ± 2.0 pS (n = 5). (C) The development of a rather high level of Piezo1 activity in the whole-cell membrane in response to 5 μM Yoda1 application. The parts of the traces indicated by arrows are shown in extended timescale. The following addition of MS channel blocker Gd3+ (10 µM GdCl3) to the bath resulted in the inhibition of Piezo1 activity. In (A) and (C), the substitutions of the extracellular solutions could clearly be seen.
Fig 4: The detection of Piezo1 activity in response to submicromolar Yoda1 concentration. Representative whole-cell recording demonstrates single Piezo1 currents at various extracellular bath Yoda1 concentrations starting from 0.5 μM. Corresponding amplitude histograms and NPo values (calculated on presented intervals) are shown below the traces.
Fig 5: Piezo1 is expressed in human leukemia K562 cells. (A) RT-PCR analysis revealed the presence of hPIEZO1 mRNA. Cropped gel with enhanced contrast is shown. (B) Immunofluorescent staining with specific antibodies detected Piezo1 proteins (Anti-Piezo1, red channel) in the cells. Cell nuclei were counterstained with DAPI (blue channel). Cells in white frame are shown in 2× zoom. No staining of the cells was observed after pre-incubation of the anti-Piezo antibody with the specific corresponding blocking peptide (Anti-Piezo+BP). (C) The single-channel activity of Piezo1 induced by Yoda1 (10 µM in the pipette solution) recorded in the representative cell-attached experiment at different membrane potentials. Here and elsewhere, the index shows a number of active channels (C—closed state (zero current), O—channel openings). Holding membrane potential is indicated near current traces. (D) The mean I-V relationship corresponds to a single-channel conductance of 19.2 ± 1.2 pS (n = 9).
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