Human Lyp Antibody

Detection of Human Lyp by Western Blot. Western blot shows lysates of Daudi human Burkitt's lymphoma cell line and Ramos human Burkitt's lymphoma cell lines. PVDF membrane was probed with 0.3 µg/mL of Goat Anti-Human Lyp Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3428) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). A specific band was detected for Lyp at approximately 108 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Lyp in Human PBMCs. Lyp was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) using Goat Anti-Human Lyp Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3428) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasmic. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Detection of Human Lyp by Simple Western^TM. Simple Western lane view shows lysates of Daudi human Burkitt's lymphoma cell line and Ramos human Burkitt's lymphoma cell line, loaded at 0.2 mg/mL. A specific band was detected for Lyp at approximately 116-118 kDa (as indicated) using 3 µg/mL of Goat Anti-Human Lyp Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3428) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Lyp by Western Blot Alternatively spliced forms of human protein tyrosine phosphatase, non-receptor type 22 (PTPN22). (D) Jurkat cells were transfected with indicated siRNA. Cell extract of the transfect cells was then analyzed on Western blotting using anti-PTPN22 and anti-Hsp90 (the left panel). Extract from 293 T cells transfected with an expression vector of Lyp2 was included in the Western blotting (Lyp2 tx 293 T). The levels of the dominant 110 kD PTPN22 protein bands were quantified with a densitometer and normalized against the level of Hsp90 from the corresponding samples, and are shown in the right panel. The normalized level of the mock-transfected cells was arbitrarily set as 100%. Statistical analysis of three independent experiments was performed with one-way ANOVA followed by Tukey’s test. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/24433447), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lyp by Western Blot Alternatively spliced forms of human protein tyrosine phosphatase, non-receptor type 22 (PTPN22). (B) 293 T cells were transfected with 1 μg of an expression vector expressing indicated FLAG-PTPN22 isoforms. The protein levels of FLAG-PTPN22 isoforms and Hsp90 in the transfected cells were determined with Western blotting. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/24433447), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Lyp by Immunocytochemistry/ Immunofluorescence LYP co-localizes with SLP76 in T cell microclusters. Jurkat cells (A) or PBL (B) were plated on coverslips covered with antibody for CD3 for the indicated periods of time. Then, cells were fixed and stained with specific antibodies for SLP76 and LYP, as indicated. Images were taken with a confocal microscope and representative images are shown. C, Wild type and 494 Jurkat cell lines were plated on coverslips covered with CD3 antibody for the indicated periods of time. Then, cells were fixed and stained with specific antibodies for SLP76. D, PBL cells were left untreated or stimulated with PHA for 72 h to induce the expression of LYP. As before, they were plated on stimulatory coverslips and processed to detect LYP and SLP76 by immunofluorescence with a confocal microscope. E, LYP expression in PLB treated with PHA. Scale bar represents 5 μm Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/39342392), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Lyp by Immunocytochemistry/ Immunofluorescence LYP co-localizes with SLP76 in T cell microclusters. Jurkat cells (A) or PBL (B) were plated on coverslips covered with antibody for CD3 for the indicated periods of time. Then, cells were fixed and stained with specific antibodies for SLP76 and LYP, as indicated. Images were taken with a confocal microscope and representative images are shown. C, Wild type and 494 Jurkat cell lines were plated on coverslips covered with CD3 antibody for the indicated periods of time. Then, cells were fixed and stained with specific antibodies for SLP76. D, PBL cells were left untreated or stimulated with PHA for 72 h to induce the expression of LYP. As before, they were plated on stimulatory coverslips and processed to detect LYP and SLP76 by immunofluorescence with a confocal microscope. E, LYP expression in PLB treated with PHA. Scale bar represents 5 μm Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/39342392), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Lyp by Western Blot Regulation of TCR signaling by LYP downstream of ZAP70. A Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells transfected with Vav and stimulated with CD3 plus CD28 antibodies for 6 h, as indicated. The insert shows the IB of the Vav and LYP proteins expressed. *P < 0.05 and **P < 0.01 for comparison of cells transfected with different plasmids and cells transfected with empty vector (pEF). B Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells transfected with rac-QL, a dominant active mutant of rac, and LYP and and stimulated with CD3 plus CD28 antibodies for 6 h. The insert shows the IB of LYP. *P < 0.05 and **P < 0.01 for comparison of cells transfected with different plasmids and cells transfected with empty vector (pEF) C Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in wild type and 494 Jurkat cells transfected with LYP and SLP76 as indicated. The cells were left untreated or stimulated with CD3/CD28 antibodies for for 6 h. The insert shows the IB of the SLP76 and LYP proteins expressed. *P < 0.05 **P < 0.01 and ***P < 0.001 for comparison of cells transfected with different plasmids and cells transfected with empty vector (pEF). D Wild type and 494 JK cells were stimulated with CD3/CD28 antibodies during the indicated periods of time. Phosphorylation of Y128 of SLP76 in each condition was measured in cell lysates by IB. Similarly, phosphorylation of LCK in Y394 and Y493 are shown. E, Wild type Jurkat cells and cells deficient in LYP (JK 494) transfected with LYP were stimulated with CD3/CD28 antibodies for 15 min and the phosphorylation of Y128 of SLP76, and Y191 of LAT were detected in each condition by IB Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/39342392), licensed under a CC-BY license. Not internally tested by R&D Systems.