anti rabbit p2y6r polyclonal antibody  (Alomone Labs)

Journal: International Journal of Molecular Sciences

Article Title: Testosterone Enhances K V Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y 4 Receptors and Adenylyl Cyclase Pathway

doi: 10.3390/ijms25094652

Figure Lengend Snippet: The agonist of P2Y receptors, UTP, generates K + currents that are amplified by testosterone (TES) via the androgen receptor signaling. ( A ) Depolarizing pulses ranging from −60 mV to +50 mV were applied to guinea pig tracheal myocytes. These pulses generated outward K + currents (IK + ) which displayed an increase in response to UTP (1 µM, 10 µM, 100 µM, and 1000 µM) in a concentration-dependent manner; n = 6. ( B ) The treatment of smooth muscle cells with TES 40 nM for 48 h enhanced the UTP-induced IK + ( n = 6), while flutamide (Flu, 3.2 µM, the androgen receptor antagonist) abolished this effect, n = 6, ( C ). Figures ( D – H ) display the statistical analysis of the individual UTP concentrations used in the different experimental groups (control without TES, TES, and Flu + TES). Notice that Flu hindered the TES-induced potentiation of IK + when they were triggered by depolarizing pulses and/or UTP. Figures ( D – H ) use different Y -axis scales to illustrate statistical significance. Note that the IK + produced by each tested UTP concentration is smaller than the IK + elicited by ATP. Symbols represent the mean values ± S.E.M. Analysis of variance followed by Dunnett’s test was used for statistical analysis. In ( D ), * p < 0.05, ** p < 0.01 comparing TES group vs. control or Flu groups; in ( E – H ), * p < 0.05, ** p < 0.01 comparing TES + UTP vs. UTP or Flu + TES + UTP groups.

Article Snippet: Then, the sections were incubated with the primary antibody anti-P2Y 4 diluted 1:75 overnight at 4 °C (Alomone Labs, APR-006, Jerusalem, Israel).

Techniques: Amplification, Generated, Concentration Assay, Control, Produced

Journal: International Journal of Molecular Sciences

Article Title: Testosterone Enhances K V Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y 4 Receptors and Adenylyl Cyclase Pathway

doi: 10.3390/ijms25094652

Figure Lengend Snippet: Role of P2Y 4 receptors in the improvement of UTP-induced K + currents caused by testosterone (TES). ( A ) Outward K + currents (IK + ) were elicited by depolarizing steps from −60 to +50 mV (control, closed circle). The IK + increased in response to 100 µM UTP. This effect was partially attenuated by 100 µM RB2, which antagonizes the action of P2Y 1,4,6,11,12,13 receptors except for P2Y 2 , and completely abolished by 10 µM AR-C118925XX, a specific P2Y 2 receptor antagonist ( n = 5). ( B ) In tracheal myocytes, UTP-induced IK + increased significantly after 48 h of exposure to 40 nM TES (open triangle). These currents were blocked by 100 µM RB2, and the subsequent addition of 10 µM AR-C118925XX did not modify the IK + ( n = 5). These results suggest that the TES-mediated enhanced response to UTP involves P2Y 4 receptors. Symbols represent mean values ± S.E.M. In figure ( A ), * p < 0.05, ** p < 0.01 comparing UTP vs. control, RB2 + UTP and RB2 + ARC + UTP groups; † p < 0.05, †† p < 0.01 comparing control vs. RB2 + UTP group. In figure ( B ), * p < 0.05, ** p < 0.01 comparing TES + UTP vs. TES, TES + RB2 + UTP and TES + RB2 + ARC + UTP groups; ¶ p < 0.05 comparing TES vs. TES + RB2 + ARC + UTP group. A one-way analysis of variance was performed, followed by a Student–Newman–Keuls test.

Article Snippet: Then, the sections were incubated with the primary antibody anti-P2Y 4 diluted 1:75 overnight at 4 °C (Alomone Labs, APR-006, Jerusalem, Israel).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Testosterone Enhances K V Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y 4 Receptors and Adenylyl Cyclase Pathway

doi: 10.3390/ijms25094652

Figure Lengend Snippet: Testosterone (TES) does not modify the expression of P2Y 4 receptors in airway smooth muscle. The first column shows preparations from guinea pig trachea, in which immunofluorescence for P2Y 4 receptors in airway muscle is shown in green without ( A ) and with 40 nM TES incubated for 48 h ( B ). ( C ) The P2Y 4 receptors did not fluoresce when no antibody was used (NC = negative control). The second column shows the α-actin of the smooth muscle (red), while the third column shows the cell nuclei (blue). Merged images from the first three panels are shown in the fourth column indicating the colocalization of P2Y 4 receptors in the smooth muscle. SM = smooth muscle, EPI = epithelium.

Article Snippet: Then, the sections were incubated with the primary antibody anti-P2Y 4 diluted 1:75 overnight at 4 °C (Alomone Labs, APR-006, Jerusalem, Israel).

Techniques: Expressing, Immunofluorescence, Incubation, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Primers for conventional polymerase chain reaction (PCR).

Article Snippet: Cells were permeabilized with 0.2% Triton X-100 in PBS-CM for 10 min at room temperature (RT) and blocked with PBS–1% bis(trimethylsilyl)acetamide (BSA) for 30 min. Then cells were incubated with primary antibodies against P2X4R, P2X7R, or P2Y 2 R (1:100) (Alomone Labs, Jerusalem, Israel) in a humidified chamber overnight at 4°C.

Techniques: Polymerase Chain Reaction, Sequencing, Amplification

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Primers for real-time quantitative PCR (qPCR).

Article Snippet: Cells were permeabilized with 0.2% Triton X-100 in PBS-CM for 10 min at room temperature (RT) and blocked with PBS–1% bis(trimethylsilyl)acetamide (BSA) for 30 min. Then cells were incubated with primary antibodies against P2X4R, P2X7R, or P2Y 2 R (1:100) (Alomone Labs, Jerusalem, Israel) in a humidified chamber overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Amplification

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Purinergic receptor pattern expression in normal and cancer-derived cell lines. (A) Representative polymerase chain reaction (PCR) from total RNA obtained from AGS (2) or GES-1 (3) cells for P2X receptors (P2XRs) (left) and P2Y receptors (P2YRs) (right). Lane 1 represents a blank control; the data shown is representative of at least three separate experiments. (B) Summary of quantitative PCR (qPCR) experiments showing RNA levels of P2X4R and P2X7R (left graph) or P2Y 1 R, P2Y 2 R, and P2Y4R (right graph) in control GES-1 cells or in gastric cancer (GC)-derived AGS, MKN-45, or MKN-74 cells. * p < 0.05, Kruskal–Wallis and Dunn´s tests. (C) Western blot analysis for the expression of P2X4R, P2X7R, P2Y 1 R, P2Y 2 R, and β-actin in HEK293, AGS, and GES-1 cells. Blots are representative of three different experiments.

Article Snippet: Cells were permeabilized with 0.2% Triton X-100 in PBS-CM for 10 min at room temperature (RT) and blocked with PBS–1% bis(trimethylsilyl)acetamide (BSA) for 30 min. Then cells were incubated with primary antibodies against P2X4R, P2X7R, or P2Y 2 R (1:100) (Alomone Labs, Jerusalem, Israel) in a humidified chamber overnight at 4°C.

Techniques: Expressing, Derivative Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Protein expression of purinergic receptors in normal and cancer-derived cell lines. (A) Confocal images from GES-1 and AGS cells showing immunofluorescence for P2Y 2 R, P2X4R, and P2X7R; scale bar 10 µm. (B) Confocal images from MKN-45 and MKN-74 cells showing immunofluorescence for P2Y 2 R and P2X4R; scale bar 10 µm. In all cases the images shown are representative of three different samples.

Article Snippet: Cells were permeabilized with 0.2% Triton X-100 in PBS-CM for 10 min at room temperature (RT) and blocked with PBS–1% bis(trimethylsilyl)acetamide (BSA) for 30 min. Then cells were incubated with primary antibodies against P2X4R, P2X7R, or P2Y 2 R (1:100) (Alomone Labs, Jerusalem, Israel) in a humidified chamber overnight at 4°C.

Techniques: Expressing, Derivative Assay, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Changes in RNA levels of P2Y 2 R and P2X4R in human biopsies from normal and tumoral gastric tissues. (A) Summary of the changes of RNA measured by qPCR for P2Y 2 R (left graph) or P2X4R (right graph) taken from seven different patients diagnosed with GC. Changes in RNA levels were measured comparing the expression of the purinergic receptor in the tumor with its levels in the adjacent healthy gastric mucosa. * p < 0.05, paired t -test. (B) Individual changes in P2Y 2 R (left) and P2X4R (right) RNA levels in the seven different tumor biopsies analyzed. Dotted line represents the basal expression in adjacent healthy gastric mucosa.

Article Snippet: Cells were permeabilized with 0.2% Triton X-100 in PBS-CM for 10 min at room temperature (RT) and blocked with PBS–1% bis(trimethylsilyl)acetamide (BSA) for 30 min. Then cells were incubated with primary antibodies against P2X4R, P2X7R, or P2Y 2 R (1:100) (Alomone Labs, Jerusalem, Israel) in a humidified chamber overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Kaplan–Meier survival plots for high or low expression of P2Y 2 R and P2X4R in GC patients. Database was taken from the Kaplan–Meier Plotter Database (kmplot.com), from a total of 593 patients diagnosed with GC. Plots show the survival of patients with low (black line) and high (red line) expression of the P2Y 2 R (left plot) or the P2X4R (right plot)—number indicates surviving patients at 0, 50, 100, and 150 months.

Article Snippet: Cells were permeabilized with 0.2% Triton X-100 in PBS-CM for 10 min at room temperature (RT) and blocked with PBS–1% bis(trimethylsilyl)acetamide (BSA) for 30 min. Then cells were incubated with primary antibodies against P2X4R, P2X7R, or P2Y 2 R (1:100) (Alomone Labs, Jerusalem, Israel) in a humidified chamber overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Primers for conventional polymerase chain reaction (PCR).

Article Snippet: Membranes were blocked with buffer [5% nonfat dry milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T)] at room temperature for 1 h. Blots were incubated with primary antibodies against P2X4R, P2X7R, P2Y 2 R, P2Y 1 R (Alomone Labs, Jerusalem, Israel), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight.

Techniques: Polymerase Chain Reaction, Sequencing, Amplification

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Primers for real-time quantitative PCR (qPCR).

Article Snippet: Membranes were blocked with buffer [5% nonfat dry milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T)] at room temperature for 1 h. Blots were incubated with primary antibodies against P2X4R, P2X7R, P2Y 2 R, P2Y 1 R (Alomone Labs, Jerusalem, Israel), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Amplification

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Purinergic receptor pattern expression in normal and cancer-derived cell lines. (A) Representative polymerase chain reaction (PCR) from total RNA obtained from AGS (2) or GES-1 (3) cells for P2X receptors (P2XRs) (left) and P2Y receptors (P2YRs) (right). Lane 1 represents a blank control; the data shown is representative of at least three separate experiments. (B) Summary of quantitative PCR (qPCR) experiments showing RNA levels of P2X4R and P2X7R (left graph) or P2Y 1 R, P2Y 2 R, and P2Y4R (right graph) in control GES-1 cells or in gastric cancer (GC)-derived AGS, MKN-45, or MKN-74 cells. * p < 0.05, Kruskal–Wallis and Dunn´s tests. (C) Western blot analysis for the expression of P2X4R, P2X7R, P2Y 1 R, P2Y 2 R, and β-actin in HEK293, AGS, and GES-1 cells. Blots are representative of three different experiments.

Article Snippet: Membranes were blocked with buffer [5% nonfat dry milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T)] at room temperature for 1 h. Blots were incubated with primary antibodies against P2X4R, P2X7R, P2Y 2 R, P2Y 1 R (Alomone Labs, Jerusalem, Israel), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight.

Techniques: Expressing, Derivative Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Protein expression of purinergic receptors in normal and cancer-derived cell lines. (A) Confocal images from GES-1 and AGS cells showing immunofluorescence for P2Y 2 R, P2X4R, and P2X7R; scale bar 10 µm. (B) Confocal images from MKN-45 and MKN-74 cells showing immunofluorescence for P2Y 2 R and P2X4R; scale bar 10 µm. In all cases the images shown are representative of three different samples.

Article Snippet: Membranes were blocked with buffer [5% nonfat dry milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T)] at room temperature for 1 h. Blots were incubated with primary antibodies against P2X4R, P2X7R, P2Y 2 R, P2Y 1 R (Alomone Labs, Jerusalem, Israel), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight.

Techniques: Expressing, Derivative Assay, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Changes in RNA levels of P2Y 2 R and P2X4R in human biopsies from normal and tumoral gastric tissues. (A) Summary of the changes of RNA measured by qPCR for P2Y 2 R (left graph) or P2X4R (right graph) taken from seven different patients diagnosed with GC. Changes in RNA levels were measured comparing the expression of the purinergic receptor in the tumor with its levels in the adjacent healthy gastric mucosa. * p < 0.05, paired t -test. (B) Individual changes in P2Y 2 R (left) and P2X4R (right) RNA levels in the seven different tumor biopsies analyzed. Dotted line represents the basal expression in adjacent healthy gastric mucosa.

Article Snippet: Membranes were blocked with buffer [5% nonfat dry milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T)] at room temperature for 1 h. Blots were incubated with primary antibodies against P2X4R, P2X7R, P2Y 2 R, P2Y 1 R (Alomone Labs, Jerusalem, Israel), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors

doi: 10.3389/fphar.2019.00612

Figure Lengend Snippet: Kaplan–Meier survival plots for high or low expression of P2Y 2 R and P2X4R in GC patients. Database was taken from the Kaplan–Meier Plotter Database (kmplot.com), from a total of 593 patients diagnosed with GC. Plots show the survival of patients with low (black line) and high (red line) expression of the P2Y 2 R (left plot) or the P2X4R (right plot)—number indicates surviving patients at 0, 50, 100, and 150 months.

Article Snippet: Membranes were blocked with buffer [5% nonfat dry milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T)] at room temperature for 1 h. Blots were incubated with primary antibodies against P2X4R, P2X7R, P2Y 2 R, P2Y 1 R (Alomone Labs, Jerusalem, Israel), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight.

Techniques: Expressing