rabbit polyclonal abs against piezo1  (Alomone Labs)

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: Store-Operated Ca 2+ Entry Contributes to Piezo1-Induced Ca 2+ Increase in Human Endometrial Stem Cells.

doi: 10.3390/ijms23073763

Figure Lengend Snippet: Figure 5. SOCE and Piezo1 in eMSCs migration. (A) Shown are representative images of the experimental wounds at the beginning (0 h) and the end of the wound healing assay (36 h). The scale bar is 200 µm (B). The eMSC wound healing dynamics in the presence of TG, Yoda1 or 2-APB in the culture media. Wound sizes are shown as mean ± SEM (n = 3–4 for each condition). * significantly different compared to control, Student’s t-test with Welch’s corrections, p < 0.05. (C) No significant changes in the viability of eMSCs were observed. Shown is the mean percent (±SD, n = 3) of dead cells for each experimental condition. #—not significantly different compared to control, two-sample Student’s t-test (p < 0.05).

Article Snippet: Then cells were incubated with goat anti-rabbit polyclonal Abs against Piezo1 (Novus Bio, Centennial, CO, USA, cat. no. NBP1-78537) or rabbit polyclonal Abs against ORAI1 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-060) or Abs against STIM1 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-063) or STIM2 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-064) at 4 ◦C overnight.

Techniques: Migration, Wound Healing Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Store-Operated Ca 2+ Entry Contributes to Piezo1-Induced Ca 2+ Increase in Human Endometrial Stem Cells

doi: 10.3390/ijms23073763

Figure Lengend Snippet: Presence of Piezo1 in eMSCs. ( A ) RT-PCR analysis revealed the presence of PIEZO1 mRNA. M—size marker, Line 1—primers specific for PIEZO1 amplified the PCR product of the expected size (360 bp), Line 2—RT-PCR negative control in which reverse transcriptase was omitted. Shown is cropped gel with enchanced contrast. ( B ) Immunofluorescent staining detected Piezo1 proteins (red channel) in eMSCs. No staining of the cells was observed after incubation of the cells with only fluorescent secondary antibodies . Cell nuclei were counterstained with DAPI (blue channel). The scale bar is 50 µm. ( C ) The single-channel activity of Piezo1 induced by selective chemical Piezo1 agonist Yoda1 (10 µM in the pipette solution). Representative current recordings at different membrane potentials are shown. Holding membrane potentials are indicated near current traces, closed and open states indicate the baseline (“zero” current) and Piezo1 open state, respectively. Note that the application of “negative” pressure ( p < 0, suction, indicated by the arrows) further increased the activity of the Yoda1-induced channels. The mean I–V relationship corresponded to single-channel conductance of 23.2 ± 1.3 pS ( n = 6).

Article Snippet: Then cells were incubated with goat anti-rabbit polyclonal Abs against Piezo1 (Novus Bio, Centennial, CO, USA, cat. no. NBP1-78537) or rabbit polyclonal Abs against ORAI1 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-060) or Abs against STIM1 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-063) or STIM2 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-064) at 4 °C overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Amplification, Negative Control, Reverse Transcription, Staining, Incubation, Activity Assay, Transferring, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Store-Operated Ca 2+ Entry Contributes to Piezo1-Induced Ca 2+ Increase in Human Endometrial Stem Cells

doi: 10.3390/ijms23073763

Figure Lengend Snippet: SOCE and Piezo1 in eMSCs migration. ( A ) Shown are representative images of the experimental wounds at the beginning (0 h) and the end of the wound healing assay (36 h). The scale bar is 200 µm ( B ). The eMSC wound healing dynamics in the presence of TG, Yoda1 or 2-APB in the culture media. Wound sizes are shown as mean ± SEM ( n = 3–4 for each condition). * significantly different compared to control, Student’s t -test with Welch’s corrections, p < 0.05. ( C ) No significant changes in the viability of eMSCs were observed. Shown is the mean percent (±SD, n = 3) of dead cells for each experimental condition. #—not significantly different compared to control, two-sample Student’s t -test ( p < 0.05).

Article Snippet: Then cells were incubated with goat anti-rabbit polyclonal Abs against Piezo1 (Novus Bio, Centennial, CO, USA, cat. no. NBP1-78537) or rabbit polyclonal Abs against ORAI1 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-060) or Abs against STIM1 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-063) or STIM2 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-064) at 4 °C overnight.

Techniques: Migration, Wound Healing Assay, Control