Journal: Cellular & Molecular Biology Letters

Article Title: GPD2 inhibition impairs coagulation function via ROS/NF-κB/P2Y12 pathway

doi: 10.1186/s11658-025-00759-x

Figure Lengend Snippet: GPD2 gene (cg03230175) methylation modulates intracellular ROS levels, which can influence P2Y12 gene expression by modulating the activation of NF-κB. NF-κB p65 binding to the P2Y12 promoter was determined by ChIP assay. A Flow cytometric analysis. The left panel shows a scatter plot of all events, highlighting P1 region. The right panel presents a histogram of fluorescence intensity for the B525-FITC-H channel within the P1 gate. B Summary flow cytometric analysis of cells from the three groups. Each panel represents the fluorescence intensity histograms for each group. C Statistical analysis bar plot of ROS levels. The ROS levels in each group are shown. Data expressed as mean ± standard error on the mean (SEM), n = 3. D Statistical chart of the relative NF-κB p65 protein levels after various interventions, accompanied by Western blot analysis. E Statistical chart of relative cytosolic NF-κB p65 protein level, accompanied by Western blot analysis. F Statistical chart of relative nuclear NF-κB p65 protein level, accompanied by Western blot analysis. G , H Statistical chart and Western blot analysis of relative levels of phosphorylated P65 (p-P65), total P65, and phosphorylated IκBα (p-IκBα) proteins. I Schematic diagram showing the NF-κB binding position in the P2Y12 promoter. J Statistical bar graph of the relative mRNA levels. K Gel electrophoresis banding pattern compares mRNA levels under input and ChIP conditions across the same three groups. The right panel shows the amplification of the P2Y12 promoter region containing the NF-κB binding motif after ChIP from MEG-01 cells, and the left panel shows the total DNA input. GAPDH was used as a control to demonstrate precipitation specificity

Article Snippet: Antibodies against GPD2, P2Y12, CD62P, VAMP8, VASP, β-actin, Histone H3, α-tubulin, NF-κB P65, p-P65, and p-IκBα were purchased from the MedChemExpress platform.

Techniques: Methylation, Gene Expression, Activation Assay, Binding Assay, Fluorescence, Western Blot, Nucleic Acid Electrophoresis, Amplification, Control

Journal: The Journal of Neuroscience

Article Title: Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue

doi: 10.1523/JNEUROSCI.0218-19.2019

Figure Lengend Snippet: Expression of P2Y12 and A2A receptors by human microglia. A, Immunostaining against P2Y12 and A2A receptors in an IBA1+, ramified PTC microglia. B, P2Y12 and A2A receptor immunostaining for an amoeboid IBA1+ cell from MTLE tissue. A, B, From left, P2Y12 immunostaining (green), IBA1 (white), A2A (red), and merged P2Y12 and A2A staining. C, Proportion of ramified (clear; n = 33) and amoeboid (hatched; n = 25) microglia immunopositive for P2Y12 (gray) and A2A receptors (red). D, Color-coded staining intensity (green represents low; red represents high) shows that P2Y12 staining was highest toward the tips of processes of a ramified microglia (above) in the resting state. Intensity was highest in a somatic region of a nonstimulated amoeboid cell (below). E, Color-coded intensity (green represents low; red represents high) for P2Y12 immunopositivity for initially amoeboid microglia fixed at full extension induced by 10 μm ADP. Staining intensity was low in perisomatic regions (top) and highest at bulbous endings of processes (bottom).

Article Snippet: We used primary antibodies directed against the following: Iba1 (Abcam, ab5076; 1/500), P2Y12 (Novus, NBP2–33870; 1/200), A2AR (Santa Cruz Biotechnology, sc-32261; 1/100), P2Y1 (Abcam, ab168918; 1/200), and P2Y13 (LSBio, LS-A1622; 1/200).

Techniques: Expressing, Immunostaining, Staining

Journal: The Journal of Neuroscience

Article Title: Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue

doi: 10.1523/JNEUROSCI.0218-19.2019

Figure Lengend Snippet: P2Y12 receptors and microglial motilities. A, The nonhydrolyzable ADP analog 2MeSADP (1 nm) increased mean microglial cross-sectional area (n = 4 PTC cells). B, The P2Y12 receptor antagonist PSB0739 (1 μm) reduced the mean cross-sectional area of ramified microglia (3 PTC and 2 MTLE microglia). It prevented the process extension induced by 10 μm ADP. C, PSB0739 suppressed surveillance motility of processes of ramified microglia. Right, Control. Left, Process positions at 5 min intervals during control period shown by colors (green, red, yellow, dark blue, light blue). D, Right, After PSB0739. Middle, Processes in the presence of PSB0739 (1 μm) and ADP (10 μm). Left, Process positions at 5 min intervals (colors as in C). Movie 4 shows that PSB0739 suppresses surveillance motility and blocks ADP-induced process extension.

Article Snippet: We used primary antibodies directed against the following: Iba1 (Abcam, ab5076; 1/500), P2Y12 (Novus, NBP2–33870; 1/200), A2AR (Santa Cruz Biotechnology, sc-32261; 1/100), P2Y1 (Abcam, ab168918; 1/200), and P2Y13 (LSBio, LS-A1622; 1/200).

Techniques: Control

Journal: The Journal of Neuroscience

Article Title: Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue

doi: 10.1523/JNEUROSCI.0218-19.2019

Figure Lengend Snippet: Role of P2Y receptors in microglial motility induced by tissue damage. A, The effects of local laser stimulation. Control images of an initially amoeboid cell from PTC before (CTRL), immediately after laser damage (yellow arrow), and at 10 and 15 min later. Merge is the difference between control and 15 min images. Red represents new membrane signal. Green represents lost signal. B, The same cell after a second laser stimulus (yellow arrow) applied 5 min after the P2Y12 antagonist PSB0739 (1 μm). Images are shown at 5 and 15 min after stimulation. Merge is the difference between PSB0739 and 15 min images. Red represents lost membrane signal due to process retraction. Blue represents new membrane signal from ruffling. Movie 6 shows that local laser damage induces process extension and is blocked by P2Y12R antagonist. Movie 7 shows that process extension is restored in the absence of P2Y12R antagonist.

Article Snippet: We used primary antibodies directed against the following: Iba1 (Abcam, ab5076; 1/500), P2Y12 (Novus, NBP2–33870; 1/200), A2AR (Santa Cruz Biotechnology, sc-32261; 1/100), P2Y1 (Abcam, ab168918; 1/200), and P2Y13 (LSBio, LS-A1622; 1/200).

Techniques: Control, Membrane

Journal: Scientific reports

Article Title: Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy.

doi: 10.1038/s41598-023-36545-y

Figure Lengend Snippet: Figure 1. P2RYs transcript levels in myoblasts derived from Tibialis anterior (TA), Gastrocnemius (GC), Soleus (SOL) and Flexor Digitorum Brevis (FDB) muscles. Transcripts encoding P2RY2, P2RY4 and P2RY6 were tested in the same sample, thus their relative expression levels can be compared quantitatively., mRNAs encoding two ADP-activated receptors (P2RY1, P2RY12) were also detected in these samples. *p < 0.05, **p < 0.01 (mdx vs. w/t).

Article Snippet: The following primary antibodies were used: P2RY1 (1:270, APR-009), P2RY2 (1:270, APR-010), P2RY4 (1:300, APR-006), P2RY6 (1:250, APR-011), P2RY12 (1:270, APR-012), P2RY13 (1:270, APR017); all Alomone Labs, calsequestrin (ab126241), calreticulin (ab128885), SERCA1 (ab124501) and SERCA2 (ab91032); all Abcam, diluted 1:1000, Gαq11 (06-709 Merck Millipore, 1:1000,), PLCβ isoforms 3–4 (sc-133231, sc-166131, respectively; Santa Cruz Biotechnology, all diluted 1:100,), NCX1 (R3F1 Swant, 1:1000), NCX3 (ab84708 Abcam, 1:500), PMCA (ab2825 Abcam, 1:1000) and IP3R (#8568 Cell Signalling Technology, 1:1000).

Techniques: Derivative Assay, Muscles, Expressing

Journal: Scientific reports

Article Title: Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy.

doi: 10.1038/s41598-023-36545-y

Figure Lengend Snippet: Figure 4. Western bloting analysis of ADP-activated metabotropic receptors P2RY12 and P2RY13. Each bar shows Western blot data from 3 independent experiments ± SD. Representative western blots are also shown. *p < 0.05 (mdx vs. w/t).

Article Snippet: The following primary antibodies were used: P2RY1 (1:270, APR-009), P2RY2 (1:270, APR-010), P2RY4 (1:300, APR-006), P2RY6 (1:250, APR-011), P2RY12 (1:270, APR-012), P2RY13 (1:270, APR017); all Alomone Labs, calsequestrin (ab126241), calreticulin (ab128885), SERCA1 (ab124501) and SERCA2 (ab91032); all Abcam, diluted 1:1000, Gαq11 (06-709 Merck Millipore, 1:1000,), PLCβ isoforms 3–4 (sc-133231, sc-166131, respectively; Santa Cruz Biotechnology, all diluted 1:100,), NCX1 (R3F1 Swant, 1:1000), NCX3 (ab84708 Abcam, 1:500), PMCA (ab2825 Abcam, 1:1000) and IP3R (#8568 Cell Signalling Technology, 1:1000).

Techniques: Western Blot