rabbit polyclonal antibody against piezo1  (Novus Biologicals)

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Protein expression levels of Cav1.2, Piezo1, CaM, and Src in human LAA tissues. (A) Representative western blots and densitometric analysis of Cav1.2 and Piezo1 proteins in LA tissues of AF patients and those with SR. (B) Representative western blots and densitometric analysis of CaM and Src protein in LA tissues of AF patients and those with SR. GAPDH was the internal control. ** p < 0.01. Values are presented as the mean ± standard error of the mean (SEM).

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effects of hypertension on the incidence of AF, I Ca,L , and Piezo1 expression in Wistar rats and SHRs with and without Val treatment. (A) Representative baseline surface ECG and intra-atrial electrocardiogram (IAEG); (B) The incidence AF in Wistar rats and SHRs treated with and without Val treatment ( n = 8). ** p < 0.01 vs. Wistar rat; ## p < < 0.01 vs. SHRs. (C) Typical surface ECG recordings of rats with AF that spontaneously reverted to SR and typical disorganized amplification of atrial waves (f wave). (D) Representative traces of AP in atrial myocytes from Wistar rats, SHRs, and SHR + Val groups and a histogram of APD in atrial myocytes from each group ( n = 12–15 myocytes from 3–4 rats). * p < 0.05 vs. Wistar rat; # p < 0.05 vs. SHRs. (E) Representative traces of I Ca,L (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in atrial myocytes of each group ( n = 8–14 myocytes from 3–4 rats). (F) Representative examples of immunohistochemical analysis of LA tissues from Wistar rats and SHRs treated with and without Val using Ab against Piezo1. Scale bar, 20 μm. (G) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in LA tissues of Wistar rats and SHRs. GAPDH was the internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Amplification, Activation Assay, Immunohistochemical staining, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of HHP on the depression of I Ca,L in HL-1 cells. (A) Representative traces of AP in HL-1 cells under various hydrostatic pressures (0, 20, and 40 mmHg) for 24 h. APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 10, and 7 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (B) Representative traces (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L ( n = 8–18 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (C) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in HL-1 cells under various hydrostatic pressure (0, 20, and 40 mmHg) for 24 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: The effects of Piezo1 on perceiving HHP and mediating the decrease of I Ca,L . (A,B) Representative Ca 2+ traces and Δ Ca i 2 + (ΔF/F) are shown. Ca 2+ entry was evoked by 10 μm Yoda1 in HL-1 cells stimulated by HHP in the presence or absence of the Piezo1 inhibitor GsmTx4 ( n = 50) or siRNA specifically knockdown Piezo1 ( n = 53–58). si-C, scrambled (control) siRNA; si-P, siRNA directed against Piezo1. (C) Representative traces of AP in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment (3.0 μM) and APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 7, and 11 at 0, 40, and 40 mmHg + GsmTx4). * p < 0.05, ** p < 0.01 vs. 0 mmHg; # p < 0.05 vs. 40 mmHg. (D) Representative traces (pulse protocol, inset), corresponding current–voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment ( n = 9–15). ** p < 0.01 vs. 0 mmHg; ## p < 0.01 vs. 40 mmHg. (E) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (F) Representative blots and densitometry analysis of Cav1.2 in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of CaM/Src on the decrease of I Ca,L induced by HHP or Yoda1 stimulation. (A) Representative blots and densitometry analysis of CaM and Src in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (B) Representative blots and densitometry analysis of CaM and Src in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. (C) Representative blots and densitometry analysis of Src and p-Src ( n = 4) in HL-1 cells transfected with scrambled (control) siRNA or siRNA directed against Piezo1 for 48 h, then treated with Yoda1 at different dosages (0, 1, and 3 μM) for 15 min. (D) Representative traces of AP and histogram of APD in HL-1 cells ( n = 7–8). * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (E) Current–voltage relationship for I Ca,L ( n = 9–17) in HL-1 cells stimulated by 40 mmHg pressure treated with 15 μM PP1 or W7. * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (F) Representative blots and densitometry analysis of Cav1.2 and Src in HL-1 cells stimulated by 40 mmHg pressure treated with W7 under different concentrations (5, 10, 15, and 20 μM). (G) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure treated with PP1 (15 μM). (H) Current-voltage relationship for I Ca,L ( n = 8–10) in HL-1 cells stimulated by Yoda1(3 μM) treated with PP1. * p < 0.05, ** p < 0.01 vs. DMSO. # p < 0.05, ## p < 0.01 vs. Yoda1. (I) Representative blots and densitometry analysis of Cav1.2 in Yoda1(3μM) -stimulated HL-1 cells treated with PP1 (15 μM). GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Transfection

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Schematic representation of the mechanism for the decrease of I Ca,L induced by HHP. Piezo1 activated by HHP depressed I Ca,L contributing to increased AF susceptibility through the CaM/Src/Pitx2 pathway.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Functional Expression of Piezo1 in Dorsal Root Ganglion (DRG) Neurons

doi: 10.3390/ijms21113834

Figure Lengend Snippet: Yoda1 induces extracellular cation influx via Piezo1 in mouse dorsal root ganglion (DRG) sensory neurons. ( A ) A representative trace of Yoda1 (10 μM, 10 s) induced an inward current in DRG neurons at a holding potential of −60 mV. ( B ) The mean peak amplitude of Yoda1 (10 μM, 10 s) induced an inward current in mouse DRG neurons ( n = 12). ( C ) Ca 2+ response induced by sequential application of Yoda1 (10 μM, 10 s) in mouse DRG neurons. ( D ) Mean normalized amplitude of sequential Yoda1-induced Ca 2+ response ( n = 43). ( E ) The Ca 2+ response in the presence or absence of extracellular Ca 2+ . Bar (gray) indicates the 0 mM CaCl 2 extracellular solution applied to mouse DRG neurons. ( F ) Mean normalized amplitude of sequential Yoda1-induced Ca 2+ response ( n = 17). High potassium chloride (50 mM) is used as the neuronal marker. All results are presented as the mean ± standard error of the mean (SEM).

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane, the blots were incubated overnight at 4 °C with a rabbit polyclonal antibody against Piezo1 (1:500, Proteintech) and a mouse monoclonal antibody against β-actin (1:1000, Sigma).

Techniques: Marker

Journal: International Journal of Molecular Sciences

Article Title: Functional Expression of Piezo1 in Dorsal Root Ganglion (DRG) Neurons

doi: 10.3390/ijms21113834

Figure Lengend Snippet: Identification of Piezo1 expression in mouse and human dorsal root ganglion (DRG) neurons. ( A ) RT-PCR showing p iezo1, piezo2, trpv1 and gapdh mRNA expression in mouse DRG and lung tissue ( piezo1 and piezo2 positive control). Primer and ultrapure water were used as negative controls. ( B ) Western blot showing Piezo1 protein expression in mouse DRG, trigeminal ganglion (TG), whole brain, and spinal cord (SC). The upper bands which are indicated by arrow represent Piezo1. Second bands (*) is nonspecific. ( C ) Single-cell RT-PCR of mouse DRG neurons showing % of piezo1 and piezo2 mRNA expressing neurons. ( D ) RT-PCR showing piezo1 gene expression in two different human’s DRG (human primer and ultrapure water were used as negative controls). ( E ) Application of Yoda1 (10 μM, 10 s)-induced Ca 2+ response in human DRG neurons. High potassium chloride (50 mM) is a neuronal marker. DRG: dorsal root ganglion; Nctrl: negative control; RT-PCR: reverse transcription-polymerase chain reaction; SC: spinal cord; TG: trigeminal ganglion; hDRG: human DRG.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane, the blots were incubated overnight at 4 °C with a rabbit polyclonal antibody against Piezo1 (1:500, Proteintech) and a mouse monoclonal antibody against β-actin (1:1000, Sigma).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Western Blot, Gene Expression, Marker, Negative Control, Reverse Transcription, Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Functional Expression of Piezo1 in Dorsal Root Ganglion (DRG) Neurons

doi: 10.3390/ijms21113834

Figure Lengend Snippet: Inhibition of Piezo1 by mechanical-ion channel inhibitors and short hairpin RNA-mediated silencing of Piezo1. ( A ) The Yoda1 (10 μM, 10 s)-induced intracellular Ca 2+ increase. The Yoda1 (10 μM, 10 s)-induced intracellular Ca 2+ increase is reversibly inhibited by ruthenium red (30 μM, N and P-type nonselective Ca 2+ channel blocker, red) ( B ), GsMTx4 (2.5 μM, selective stretch-activated channel blocker, blue) ( C ), and Dooku1 (10 μM, Yoda1 analogue, green) ( D ). The arrows indicate spontaneous intracellular Ca 2+ increase. ( E ) The bars indicate treatment with various inhibitors: ruthenium red (RR) (control, n = 6; RR, n = 12, two-way ANOVA (Fisher’s LSD test), *, p < 0.05); GsMTx4 (control, n = 23; Gs, n = 22, two-way ANOVA (Fisher’s LSD test), ***, p < 0.001); and Dooku1 (control, n = 9; Dk, n = 19, two-way ANOVA (Fisher’s LSD test), *, p < 0.05). ( F ) piezo1 mRNA levels, normalized by gapdh, were quantified by a TaqMan assay after transfection with sh-piezo1 dorsal root ganglion (DRG) neurons (unpaired t-test, **** p < 0.0001). ( G ) Application of Yoda1 (10 μM, 10 s)-induced Ca 2+ response in mouse DRG neurons transfected with sh-piezo1 (red) and scramble (gray) or control (black). ( H ) Mean normalized amplitude of Yoda1-induced Ca 2+ response (control, n = 29; scramble, n = 22; sh-piezo1, n = 18, one-way ANOVA (Dunn’s multiple comparisons test, ns; nonsignificant, **, p < 0.01). High potassium chloride (50 mM) is a neuronal marker. All results are presented as the mean ± standard error of the mean (SEM) (control versus sh-piezo1 and scramble or control versus each drug). DRG: dorsal root ganglion; sh: short hairpin.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane, the blots were incubated overnight at 4 °C with a rabbit polyclonal antibody against Piezo1 (1:500, Proteintech) and a mouse monoclonal antibody against β-actin (1:1000, Sigma).

Techniques: Inhibition, shRNA, Control, TaqMan Assay, Transfection, Marker

Journal: International Journal of Molecular Sciences

Article Title: Functional Expression of Piezo1 in Dorsal Root Ganglion (DRG) Neurons

doi: 10.3390/ijms21113834

Figure Lengend Snippet: Regulation of Piezo1 by TRPV1 activation in the same dorsal root ganglion (DRG) neurons. ( A ) Representative trace of the inward current induced by Yoda1 (10 μΜ, 10 s) and capsaicin (cap, 100 nM, 10 s) in the same neuron. ( B ) The number of neurons activated by Yoda1 or/and capsaicin. ( C ) A representative trace of the Yoda1 (10 μM, 10 s)-induced inward current (control group). ( D ) A representative trace of the effect of the inward current of Piezo1 by capsaicin (cap, 100 nM, 10 s) (capsaicin treatment group). ( E ) The mean peak amplitude of the second application of Yoda1 (10 μM, 10 s) after capsaicin treatment ( D ) or not ( C ) ( n = 12, unpaired t-test, ****, p < 0.0001). ( F ) The percentage of the second Yoda1-induced amplitude after capsaicin treatment (or not) compared to the first Yoda1-induced amplitude ( C ) and ( D ) ( n = 11, unpaired t-test, ****, p < 0.0001). The results are presented as the mean ± standard error of the mean (SEM).

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane, the blots were incubated overnight at 4 °C with a rabbit polyclonal antibody against Piezo1 (1:500, Proteintech) and a mouse monoclonal antibody against β-actin (1:1000, Sigma).

Techniques: Activation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Functional Expression of Piezo1 in Dorsal Root Ganglion (DRG) Neurons

doi: 10.3390/ijms21113834

Figure Lengend Snippet: Primer information for standard RT-PCR and scRT-PCR amplification.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane, the blots were incubated overnight at 4 °C with a rabbit polyclonal antibody against Piezo1 (1:500, Proteintech) and a mouse monoclonal antibody against β-actin (1:1000, Sigma).

Techniques: Amplification