Human Phospho-SYK (Y525/Y526) Antibody

Detection of Human Phospho-SYK (Y525/Y526) by Western Blot. Western blot shows lysates of Ramos human Burkitt's lymphoma cell line untreated (-) or treated (+) with 10 µg/mL IgM for 2 minutes and U937 human histiocytic lymphoma cell line untreated (-) or treated (+) with 2nM pervanadate (PV) for 5 minutes. PVDF Membrane was probed with 0.1 µg/mL of Human Phospho-SYK (Y525/Y526) Monoclonal Antibody (Catalog # MAB6459) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for Phopho-SYK (Y525/Y526) at approximately 80 kDa (as indicated). For additional reference, the membrane was stripped and reprobed with mouse anti-SYK (lower panel). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Phospho-SYK (Y525/Y526) by Western Blot Cell permeable pharmacological inhibitor of AC enzyme activity reverses toxin-mediated inhibition of uptake of opsonized particles and associated signal transduction pathways. THP-1 human monocytes were preincubated with adefovir dipivoxil (10 µM) for 6 h, followed by incubation with ET for 6 h (green), CyaA for 30 min (red), or buffer (blue) as mentioned in the Materials and Methods section. (A and B) 2 × 105 THP-1 cells were incubated with fluorescently labelled SOZ particles for 30 min, and binding and internalization of fluorescent particles were analyzed by flow cytometry. Representative histogram from one experiment (A) and the calculated phagocytic index (B) are shown. Data represent mean with SEM (N = 4). p values were determined by paired one-way ANOVA; ns, not significant for results compared with adefovir-treated cells incubated with SOZ particles. (C to E) 3 × 106 THP-1 cells were incubated with SOZ particles to induce tyrosine phosphorylation of crucial signaling proteins involved in opsonophagocytosis. The cells were lysed, and the lysates were used for immunoblotting. Tyrosine phosphorylation of Syk (C), Pyk2 (D), and Vav (E) were detected using phospho-specific antibodies. Immunoblots developed using anti-Syk, anti-Pyk2, and anti-Vav antibodies served as loading controls. THP-1 cells preincubated without (black) or with (grey) adefovir and then treated with unopsonized zymosan were used as negative controls. Data represent mean with SEM (N = 3). p values were determined using one-way ANOVA; *, p < 0.05; **, p < 0.005; ns, not significant, for results compared with adefovir-treated cells incubated with SOZ particles. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31226835), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-SYK (Y525/Y526) by Western Blot Preincubation of THP-1 cells with adefovir dipivoxil does not affect the opsonin induced tyrosine phosphorylation of Syk, Pyk2, and Vav. The experiment was carried out as mentioned in the legend for Figure 3. Data represent mean with SEM (N = 3). p values were determined by one-way ANOVA; *** p < 0.001; **** p < 0.0001; ns, not significant, for results compared with buffer-treated cells incubated with SOZ particles. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31226835), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-SYK (Y525/Y526) by Western Blot Toxin-provoked cAMP accumulation inhibits opsonin-induced tyrosine phosphorylation of cellular proteins Syk, Pyk2, and Vav. 3 × 106 THP-1 human monocytes were preincubated with ET for 6 h (green), CyaA for 30 min (red), or buffer (blue) and subsequently incubated with SOZ particles (30 min) at 37 °C to induce tyrosine phosphorylation of crucial signaling proteins leading to opsonophagocytosis. THP-1 cells preincubated with buffer and then treated with unopsonized zymosan were taken as negative control (black). Cell lysates were analyzed by immunoblotting. (A) Modulated SOZ-induced tyrosine phosphorylation of proteins was detected in cellular lysates from toxin/buffer pretreated cells (red arrows); tubulin was used as loading control. Tyrosine phosphorylation of Syk (B), Pyk2 (C), and Vav (D) was detected using phospho-specific antibodies. Immunoblots developed with anti-Syk, anti-Pyk2, and anti-Vav antibodies served as loading controls. Data represent mean with SEM (N = 3). p values were determined using one-way ANOVA; **, p < 0.005; *** p < 0.001; **** p < 0.0001; ns, not significant, for results compared with buffer-treated cells incubated with SOZ particles. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31226835), licensed under a CC-BY license. Not internally tested by R&D Systems.