rabbit polyclonal anti p2y2r antibody  (Alomone Labs)

Journal: Frontiers in Immunology

Article Title: P2Y 2 -P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis

doi: 10.3389/fimmu.2023.1328897

Figure Lengend Snippet: Endothelial P2Y 2 R mediates the mononuclear cell adhesion induced by UTP 100 µM, and its expression is not altered by schistosomiasis. (A, B) The selective P2Y 2 R antagonist ARC118925XX (10 µM; 30 min pretreatment) inhibited mononuclear cell adhesion to endothelial cells from the control (white bars) and infected (gray bars) groups. (C, D) The NTPDase (CD39) inhibition by ARL 67156 (100 µM; 30 min pretreatment) did not alter the UTP-induced mononuclear cell adhesion effect. *** p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 (A–D) . (E) Immunoblot of P2Y 2 R in both groups. (F) Densitometric analysis of immunoblot data (a.u.). (G) P2Y 2 R mRNA levels were normalized by the endogenous GAPDH gene. Data were expressed as mean and SEM of n independent cultures for each condition. p = 0.82 and 0.12, Student’s t -test, n = 3 (F , G) .

Article Snippet: Next, the membranes were washed three times for 5 min with 1× TBS-Tween solution and incubated overnight with 1:200 rabbit anti-P2Y 2 receptor antibody (APR-010; Alomone Labs, Israel) or 1:5,000 mouse monoclonal anti-β-actin (A1978; Sigma - Aldrich, USA) diluted in 5% BSA 1× TBS-Tween solution.

Techniques: Expressing, Control, Infection, Inhibition, Western Blot

Journal: Frontiers in Immunology

Article Title: P2Y 2 -P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis

doi: 10.3389/fimmu.2023.1328897

Figure Lengend Snippet: The increased role of the P2Y 2 R canonical signaling and endothelial adhesion molecules in the UTP-induced mononuclear cell adhesion in the schistosomiasis group. White bars (left), control; gray bars (right), infected group. (A, B) The endothelial cell pretreatment (30 min) with intracellular Ca 2+ inhibitors U73122 (1 µM) or BAPTA-AM (3 µM) blocked the UTP-induced mononuclear cell adhesion and reduced basal adhesion values in the infected group. (C, D) Maximal increase of endothelial concentration of Ca 2+ in response to 100 µM ATP ( C , control = 1.56 a.u. ± 0.08 a.u.; D , infected = 1.88 a.u. ± 0.06 a.u., p < 0.05). Insert typical register of time-lapse variation of intracellular Ca 2+ in endothelial cells in response to ATP. Light line, control group; dark line, infected group. (E, F) Endothelial cell preincubation with monoclonal VCAM-1 or ICAM-1 antibodies (30 min) blocked the UTP-induced mononuclear cell adhesion. In the infected group (F) , VCAM-1 antibody also reduced the basal mononuclear cell adhesion. Data were expressed as the mean and SEM of n independent cultures for each condition. *** p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test, n = 3–4 (A , B) . *** p < 0.001 ( n = 10–18 replicates from three cultures for each condition, Student’s test) (C , D) . *** p < 0.001, * p < 0.05 (one-way ANOVA followed by Tukey’s multiple comparisons test, n = 3) (E , F) . U73122, phospholipase C inhibitor; BAPTA-AM, Ca 2+ chelator; anti-VCAM-1, vascular cell adhesion molecule antibody (dilution 1:50); anti-ICAM-1, intercellular cell adhesion molecule antibody (dilution 1:50).

Article Snippet: Next, the membranes were washed three times for 5 min with 1× TBS-Tween solution and incubated overnight with 1:200 rabbit anti-P2Y 2 receptor antibody (APR-010; Alomone Labs, Israel) or 1:5,000 mouse monoclonal anti-β-actin (A1978; Sigma - Aldrich, USA) diluted in 5% BSA 1× TBS-Tween solution.

Techniques: Control, Infection, Concentration Assay

Journal: Frontiers in Immunology

Article Title: P2Y 2 -P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis

doi: 10.3389/fimmu.2023.1328897

Figure Lengend Snippet: Evidence of endothelial P2Y 2 R and P2X7R cooperation increasing mononuclear cell adhesion to endothelial monolayer in the infected group. The selective P2X7R antagonist A740003 (50 nM, 30 min pretreatment) blocked 500 µM-induced mononuclear cell adhesion in control ( A , white bars) and infected groups ( B , gray bars). In the control group, we observed similar effects of UTP 100 µM (P2Y 2 R), ATP 500 µM (P2X7R), or the combination of both agonists (C) . In the infected group, P2Y 2 R and P2X7R coactivation with 100 µM UTP plus 500 µM ATP induced a higher mononuclear cell adhesion than each agonist alone (D) . Data were expressed as the mean and SEM of n independent cultures for each condition. *** p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 or 4). A74, A740003 (selective P2X7R antagonist).

Article Snippet: Next, the membranes were washed three times for 5 min with 1× TBS-Tween solution and incubated overnight with 1:200 rabbit anti-P2Y 2 receptor antibody (APR-010; Alomone Labs, Israel) or 1:5,000 mouse monoclonal anti-β-actin (A1978; Sigma - Aldrich, USA) diluted in 5% BSA 1× TBS-Tween solution.

Techniques: Infection, Control

Journal: Frontiers in Immunology

Article Title: P2Y 2 -P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis

doi: 10.3389/fimmu.2023.1328897

Figure Lengend Snippet: Endothelial VCAM-1 expression are selectively upregulated by the P2Y 2 R and P2X7R coactivation in the infected group. Immunocytochemistry staining of cultured endothelial cells using antibodies raised against VCAM-1 (green) or ICAM-1 (green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 µm; ×400). Basal: endothelial cells were incubated with DMEM supplemented with 0.2% SFB for 5 h (A , D) . Endothelial cells were incubated with UTP 100 µM (5 h, B , E ) or UTP 100 µM (5 h) plus ATP 500 µM (10 min) diluted in DMEM supplemented with 0.2% SFB (C , F) . Representative images from the control (A – C) and infected groups (D – F) were randomly chosen. Similar results were obtained in other experiments ( n = 4 for each condition). The fluorescence intensity was quantified for the control (white bars, G ) and infected groups (gray bars, H) and expressed as arbitrary units (a.u.). Data were expressed as the mean and SEM of n independent cultures for each condition. *** p < 0.001, ** p < 0.01, * p < 0.05 (one-way ANOVA followed by Tukey’s multiple comparisons test, n = 4) (G, H) .

Article Snippet: Next, the membranes were washed three times for 5 min with 1× TBS-Tween solution and incubated overnight with 1:200 rabbit anti-P2Y 2 receptor antibody (APR-010; Alomone Labs, Israel) or 1:5,000 mouse monoclonal anti-β-actin (A1978; Sigma - Aldrich, USA) diluted in 5% BSA 1× TBS-Tween solution.

Techniques: Expressing, Infection, Immunocytochemistry, Staining, Cell Culture, Fluorescence, Incubation, Control

Journal: Frontiers in Immunology

Article Title: P2Y 2 -P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis

doi: 10.3389/fimmu.2023.1328897

Figure Lengend Snippet: Evidence of endothelial P2Y 2 R and P2X7R cooperation increasing IL-1β release and mononuclear cell adhesion in the infected group. White bars, control; gray bars, infected group. P2Y 2 R and P2X7R activation with UTP 100 µM and ATP 500 µM, respectively, increased IL-1β release only in the infected group (A) . In this group, the coactivation of both P2 receptors induced a higher effect than the activation of each receptor individually (A) . The caspase inhibitor (Z-VAD-FMK 20 µM) blocked the UTP plus ATP-induced IL-1β release by endothelial cells (A) and the mononuclear cell adhesion only in the infected group (B , C) . The IL-1β-induced mononuclear cell adhesion was blocked by endothelial preincubation with anti-VCAM-1 antibody (dilution 1:50) or NF-κB inhibitor PDTC 3 µM, but not by anti-ICAM-1 antibody (dilution 1:50) (D, E) . Moreover, in the infected group, both the VCAM-1 antibody and PDTC also reduced basal values of mononuclear cell adhesion (E) . PDTC prevented the mononuclear cell adhesion induced by UTP, ATP, or UTP plus ATP only in the infected group (F , G) . Data were expressed as the mean and SEM of n independent cultures for each condition. *** p < 0.001, * p < 0.05 (one-way ANOVA followed by Tukey’s multiple comparisons test, n = 3–6 (A) . *** p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 ( B–G ).

Article Snippet: Next, the membranes were washed three times for 5 min with 1× TBS-Tween solution and incubated overnight with 1:200 rabbit anti-P2Y 2 receptor antibody (APR-010; Alomone Labs, Israel) or 1:5,000 mouse monoclonal anti-β-actin (A1978; Sigma - Aldrich, USA) diluted in 5% BSA 1× TBS-Tween solution.

Techniques: Infection, Control, Activation Assay

Journal: Frontiers in Immunology

Article Title: P2Y 2 -P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis

doi: 10.3389/fimmu.2023.1328897

Figure Lengend Snippet: Positive cooperation between endothelial P2Y 2 R and P2X7R signaling favoring mononuclear cell adhesion during schistosomiasis. Schistosoma eggs adhere to endothelial cells and extravasate to the peritoneum or intestinal lumen, triggering a granulomatous response. The coactivation of endothelial P2Y 2 R-P2X7Rs increases IL-1β release and VCAM-1 expression. The increased endothelial Ca 2+ canonical signaling together with ROS promotes NF-κB signaling, favoring IL-1β release, VCAM-1 expression, mononuclear cell adhesion, and transmigration. Created with BioRender.com .

Article Snippet: Next, the membranes were washed three times for 5 min with 1× TBS-Tween solution and incubated overnight with 1:200 rabbit anti-P2Y 2 receptor antibody (APR-010; Alomone Labs, Israel) or 1:5,000 mouse monoclonal anti-β-actin (A1978; Sigma - Aldrich, USA) diluted in 5% BSA 1× TBS-Tween solution.

Techniques: Expressing, Transmigration Assay