anti-p2y4 receptor antibody  (Alomone Labs)

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A , diagram showing a cross‐section side‐view of a pre‐hearing (left) and mature (right) organ of Corti. IHC: inner hair cell; OHC: outer hair cell; IPhC: inner phalangeal cell; IBC: inner border cell; PC: pillar cell; GER: greater epithelial ridge; IS: inner sulcus; SC: supporting cells of the GER and IS, which also include IPhCs and IBCs. The ‘Immature’ and ‘Mature’ designations refer to before and after the onset or hearing, respectively, which in mice occurs at around P12. The images below represent either the top view (red arrow) or the side view of the GER and IS. The image on the left was modified from Ceriani et al. . B and C , brightfield images of the organ of Corti in P7 (top), 1‐month‐old (middle) and aged (bottom) 6N ( B ) and C3H ( C ) mice taken from the top‐view orientation used for the fluorescence images in panels D and E ; these images highlight the location of the different supporting cell types (SC) in the GER and IS and the IHCs. Note that for simplicity, only a few SCs are highlighted in panels B and C . Scale bars are 20 μm. D and E , maximum intensity projections of confocal z ‐stacks showing images of the GER and IS viewed from the top (red arrow in panel A ) in P7 (top panels), 1‐month‐old (middle panels) and aged (17–21 months: bottom panels) 6N ( D ) and C3H ( E ) mice. Left columns show the actin‐marker phalloidin (magenta), which is labelling the hair bundles of the IHCs (yellow arrows) and the membrane of some of the supporting cells. Middle columns show the P2Y 1 puncta‐like labelling (white) in the supporting cells from the bulk of the GER and IS (white arrows). P2Y 1 was also expressed in the pillar cells (PCs: orange arrows) which were not investigated in this study. Right panels show the merged images. Scale bars are 20 μm. F and G , maximum intensity projections of confocal z‐stack images of the GER and IS viewed from the side (see panel A ). Phalloidin: magenta; P2Y 1 : white. Note that phalloidin primarily labels the hair bundle of the IHCs (yellow circles) and the pillar cells (PC). Scale bars are 10 μm.

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques: Modification, Fluorescence, Marker, Membrane

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A and B , maximum intensity projections of confocal z ‐stacks showing images of the GER and IS viewed from the top (red arrow in panel 2 A ) in P7 (top panels), 1‐month‐old (middle panels) and aged (17–21 months: bottom panels) 6N ( A ) and C3H ( B ) mice. Left column: actin‐marker phalloidin (magenta); middle column: P2Y 4 (white). Right panels show the merged images. For the identification of the cellular organization and labels, see Fig. . Scale bars are 20 μm. C and D , maximum intensity projections of confocal z ‐stack images of the GER and IS viewed from the side (see Fig. A ). Phalloidin: magenta; P2Y 4 : white. Scale bars are 10 μm.

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques: Marker

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A and B , maximum intensity projections of confocal z ‐stacks showing images of the GER and IS viewed from the top (red arrow in panel 2 A ) in P7 (top panels), 1‐month‐old (middle panels) and aged (17–21 months: bottom panels) 6N ( A ) and C3H ( B ) mice. Left column: actin‐marker phalloidin (magenta); middle column: P2Y (white). Right panels show the merged images. For the identification of the cellular organisation and labels, see Fig. . Scale bars are 20 μm. C and D , maximum intensity projections of confocal z ‐stack images of the GER and IS viewed from the side (see Fig. A ). Phalloidin: magenta; P2Y 4 : white. Scale bars are 10 μm.

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques: Marker

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A–C , representative Ca 2+ responses in supporting cells induced by the extracellular application of 1 μm ADP (grey area) in 6N mice at different age ranges shown above the recordings. D , comparison of the average Ca 2+ response at the onset of ADP application (grey bar beneath the traces) in cochlear supporting cells of 6N mice in the three different age ranges tested. Continuous traces represent averages, while the shaded area is the SD. Numbers of individual supporting cells (ROIs): P7–P8, 59 ROIs (3 mice); 1–2 months old, 70 ROIs (4), and 18–24 months old, 121 ROIs (5). E and F , comparison of the maximum ( E ) and average ( F ) Ca 2+ response to 1 μm ADP application in 6N mice at different ages. Number of supporting cells used is shown above the averages (± SD) and single data points (plotted as open circles). G , representative Ca 2+ responses in supporting cells induced by 1 μm extracellular ADP (grey area) in aged 6N mice. The application of ADP together with the P2Y 1 antagonist MRS2500 (1 μm, top black horizontal line) blocked the ADP‐induced Ca 2+ response. H , effect of P2Y 1 antagonist MRS2500 on the size of the ADP‐induced Ca 2+ response in cochlear supporting cells from aged mice from 64 supporting cells (ROIs) from 3 mice. Significance values are indicated by the asterisks ( P < 0.0001, Wilcoxon signed‐rank test).

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques: Comparison

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A , representative Ca 2+ responses in supporting cells induced by the extracellular application of 300 n m UTP (grey area) in aged 6N mice. The application of UTP together with the P2Y 2 antagonist ARC‐118925XX (15 μm, top black horizontal line) caused a reduction of the UTP‐induced Ca 2+ response and stopped Ca 2+ oscillations. B , average UTP‐induced Ca 2+ responses during the last 10 s of UTP application and when UTP was applied together with the P2Y 2 antagonist ARC‐118925XX in cochlear supporting cells from aged mice (74 ROIs from 4 mice). Significance values are indicated by the asterisks ( P < 0.0001, Wilcoxon signed‐rank test). C , representative Ca 2+ responses in supporting cells induced by the P2Y 4 agonist MRS4062 (10 μm, top black horizontal line) in aged 6N mice. This initial response was followed by the application of 300 n m UTP alone (grey area), to confirm that the supporting cell was responsive to UTP. D , average and maximum MRS4062‐induced Ca 2+ response in cochlear supporting cells from aged mice. Open symbols are measurements from individual supporting cells (ROIs): 84 from 3 mice.

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques:

Journal: Frontiers in Immunology

Article Title: SARS-CoV-2 Spike protein alters microglial purinergic signaling

doi: 10.3389/fimmu.2023.1158460

Figure Lengend Snippet: SARS−CoV−2 spike protein increases ATP secretion and purinergic signaling components transcript levels in microglial cells. BV-2 cells were left untreated (Ctrl) or stimulated with 0.5 or 1 µg/mL SARS−CoV−2 spike protein for 2h for ATP quantification in culture supernatants or 24 h for qPCRs. (A) ATP concentration in cell supernatants. The levels of transcripts for (B) P2X7, (C) P2X4, (D) P2Y 1 , (E) P2Y 2 , (F) P2Y 4 , (G) P2Y 6 , (H) P2Y 12 , (I) E-NTPDase1, (J) E-NTPDase2, (K) E-NTPDase3, and (L) CD73 were analyzed by RT-qPCR. Data are representative of three independent experiments (n=3) performed in triplicates and expressed as mean ± SEM. Statistically significant differences between Ctrl and treated groups are represented by asterisks (*, p < 0.05). One-way analysis of variance, Tukey’s test.

Article Snippet: Samples were then incubated overnight with the following primary antibodies diluted in 0.1% BSA in PBS: rabbit anti-P2X7 #APR-008 (1:100), rabbit anti-P2Y 1 #APR-009 (1:100), rabbit anti-P2Y 4 #APR-006 (1:300), rabbit anti-P2Y 6 #APR-011 (1:500), rabbit anti-P2Y 12 #APR-012 (1:50) (Alomone Labs, Jerusalem, Israel), goat anti-CD39 #AF4398 (1:200) (R&D Systems, Minneapolis, MN), rabbit ENTPD2/CD39L1 # BS-11515R (1:100) (Thermo Fisher Scientific, NJ, USA).

Techniques: Concentration Assay, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: SARS-CoV-2 Spike protein alters microglial purinergic signaling

doi: 10.3389/fimmu.2023.1158460

Figure Lengend Snippet: SARS−CoV−2 spike protein increases the expression of P2 receptors and ectonucleotidases in microglial cells. BV-2 cells were left untreated (Ctrl) or stimulated with 1 µg/mL SARS−CoV−2 spike protein for 24 h. Representative images and quantitative analysis for (A–C) P2X7, (D–F) P2Y 1 , (G–I) P2Y 4 , (J–L) P2Y 6 , (M–O) P2Y 12 , E-NTPDase1 (P–R) , and E-NTPDase2 (S–U) . Data are representative of three independent experiments (n=3) and expressed as mean ± SEM of 10 fields per condition. (V) ATP hydrolysis assay. Data are representative of three independent experiments (n=3) and expressed as mean ± SEM. Statistically significant differences between Ctrl and treated groups are represented by asterisks (*p < 0.05; **p < 0.01; ****p < 0.0001). Student’s t-test. Scale bars: 20 μm.

Article Snippet: Samples were then incubated overnight with the following primary antibodies diluted in 0.1% BSA in PBS: rabbit anti-P2X7 #APR-008 (1:100), rabbit anti-P2Y 1 #APR-009 (1:100), rabbit anti-P2Y 4 #APR-006 (1:300), rabbit anti-P2Y 6 #APR-011 (1:500), rabbit anti-P2Y 12 #APR-012 (1:50) (Alomone Labs, Jerusalem, Israel), goat anti-CD39 #AF4398 (1:200) (R&D Systems, Minneapolis, MN), rabbit ENTPD2/CD39L1 # BS-11515R (1:100) (Thermo Fisher Scientific, NJ, USA).

Techniques: Expressing, Hydrolysis Assay

Journal: Frontiers in Immunology

Article Title: SARS-CoV-2 Spike protein alters microglial purinergic signaling

doi: 10.3389/fimmu.2023.1158460

Figure Lengend Snippet: SARS−CoV−2 spike protein increases purinergic signaling components transcript levels in mice hippocampus. The levels of transcripts for (A) P2X7, (B) P2X4, (C) P2Y 1 , (D) P2Y 2 , (E) P2Y 4 , (F) P2Y 6 , (G) P2Y 12 , (H) E-NTPDase1, (I) E-NTPDase2, (J) E-NTPDase3, and (K) CD73 in hippocampus of mice post-spike ICV injection were analyzed by RT-qPCR. Data are expressed as mean ± SEM. Statistically significant differences between the control (Ctrl) and treated group are represented by asterisks (*, p < 0.05). Outliers were identified and excluded using Prism 8.0.1 software (GraphPad Software, La Jolla, CA, USA). Student’s t-test (Control group n = 3; Spike group n = 5–7).

Article Snippet: Samples were then incubated overnight with the following primary antibodies diluted in 0.1% BSA in PBS: rabbit anti-P2X7 #APR-008 (1:100), rabbit anti-P2Y 1 #APR-009 (1:100), rabbit anti-P2Y 4 #APR-006 (1:300), rabbit anti-P2Y 6 #APR-011 (1:500), rabbit anti-P2Y 12 #APR-012 (1:50) (Alomone Labs, Jerusalem, Israel), goat anti-CD39 #AF4398 (1:200) (R&D Systems, Minneapolis, MN), rabbit ENTPD2/CD39L1 # BS-11515R (1:100) (Thermo Fisher Scientific, NJ, USA).

Techniques: Injection, Quantitative RT-PCR, Control, Software