plasmid vector prset b  (Thermo Fisher)

Journal: The Journal of Biological Chemistry

Article Title: Phagocytosis mediated by the human granulocyte receptor CEACAM3 is limited by the receptor-type protein tyrosine phosphatase PTPRJ

doi: 10.1016/j.jbc.2022.102269

Figure Lengend Snippet: Overexpression of active PTPRJ diminishes CEACAM3-mediated phagocytosis. A, schematic representation of myristoylated (myr-) protein phosphatase domains lacking the extracellular and the transmembrane domain. PTPRS, PTPRF, and PTPRG harbor, in addition to the catalytically active phosphatase domain ( dark gray ), a second catalytically inactive domain ( light gray ). B, 293T cells were cotransfected to express CEACAM3-GFP together with the indicated phosphatase domains fused to mCherry. Equal CEACAM3-GFP expression levels and expression of the different myr-phosphatase-mCherry constructs were analyzed by Western blotting using α-GFP ( upper panel ) and α-mCherry ( middle panel ) antibodies. Probing of the samples with α-vinculin ( lower panel ) served as loading control. C, cells from (B) were infected with Opa 52 -expressing (Opa 52 ) or Opa-negative (Opa-) N. gonorrhoeae (MOI 30) for 45 min. Internalized bacteria were enumerated by gentamicin protection assays. Bars show mean ± SEM of three independent experiments performed in triplicates. n.s., not significant; ∗∗∗ p < 0.001. D, 293T cells were cotransfected with plasmids encoding GFP-tagged CEACAM3 or GFP alone together with the indicated mScarlet-tagged myristoylated phosphatase domain or mScarlet alone. Cells were infected with PacificBlue-labeled Opa 52 -expressing N. gonorrhoeae for 30 min, fixed, and analyzed by confocal microscopy. Insets show 3× magnification of boxed areas. Bars represent 10 μm (corresponding to 3.3 μm for insets). CEACAM3, carcinoembryonic antigen-related cell adhesion molecule 3; PTPR, receptor-type protein tyrosine phosphatase.

Article Snippet: The resulting expression constructs encode the respective myristoylated phosphatase domain with a fluorescent protein fused to the carboxy-terminus. pLPS3′-mScarlet and pLPS3′-mCherry were created by replacing the EGFP-encoding cDNA of the pLPS3′-EGFP acceptor vector (Clontech) with the cDNA of mScarlet derived from vector pmScarlet-C1 (kind gift from Dorus Gadella; Addgene #85042) or with the cDNA of mCherry derived from vector pRSET-B mCherry (kind gift from Kalina Hristova; Addgene #108857), respectively.

Techniques: Over Expression, Expressing, Construct, Western Blot, Control, Infection, Bacteria, Labeling, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Phagocytosis mediated by the human granulocyte receptor CEACAM3 is limited by the receptor-type protein tyrosine phosphatase PTPRJ

doi: 10.1016/j.jbc.2022.102269

Figure Lengend Snippet: PTPRJ diminishes CEACAM3 tyrosine phosphorylation. A, 293T cells were transfected to express CEACAM3-GFP or GFP. Where indicated, cells were additionally transfected with constructs encoding the mCherry-tagged myristoylated phosphatase domain of PTPRJ or the inactive mutant (PTPRJ-D/A). Transfected cells were infected with Opa 52 -expressing (Opa 52 ) or Opa-negative (Opa-) N. gonorrhoeae (MOI of 30) for 15 min or remained uninfected. Subsequently, WCL were prepared, and expression of PTPRJ-mCherry ( upper panel ) and CEACAM3-GFP or GFP ( middle panel ) was confirmed by immunoblotting with appropriate antibodies. Lysates were probed with α-tubulin antibodies to confirm equal loading (lower panel). B, lysates from ( A ) were incubated with GFP-binding nanobodies and GFP or CEACAM3-GFP was immunoprecipitated. Tyrosine phosphorylation of CEACAM3 in the precipitates was analyzed by Western blotting with α-phospho-tyrosine (pTyr) antibody ( upper panel ). Equal immunoprecipitation from the samples was verified by probing with α-CEACAM3 ( middle panel ) and α-GFP antibodies ( lower panel ). C, c-Src was immunoprecipitated from cell lysates as in ( A ). The tyrosine phosphorylation status of c-Src was determined by Western blotting with antibodies directed against phosphorylated Y418 (pY418; upper panel ; corresponds to pY419 in human c-Src) or pY527 (middle panel; corresponds to pY530 in human c-Src). Probing with α-c-Src antibodies confirmed equal loading ( lower panel ). D, c-Src was immunoprecipitated from lysates of serum-starved (-) or serum-stimulated (+) murine embryonic fibroblasts, and the samples were probed as in C). E, the tyrosine phosphorylation status of p130 Cas was determined by Western blotting with antibodies directed against phosphorylated Y410 of p130 Cas (pY410; upper panel ), while probing with α-p130 Cas antibody ( middle panel ) and α-tubulin ( lower panel ) confirmed equal loading. CEACAM3, carcinoembryonic antigen-related cell adhesion molecule 3; PTPR, receptor-type protein tyrosine phosphatase; WCL, whole cell lysate.

Article Snippet: The resulting expression constructs encode the respective myristoylated phosphatase domain with a fluorescent protein fused to the carboxy-terminus. pLPS3′-mScarlet and pLPS3′-mCherry were created by replacing the EGFP-encoding cDNA of the pLPS3′-EGFP acceptor vector (Clontech) with the cDNA of mScarlet derived from vector pmScarlet-C1 (kind gift from Dorus Gadella; Addgene #85042) or with the cDNA of mCherry derived from vector pRSET-B mCherry (kind gift from Kalina Hristova; Addgene #108857), respectively.

Techniques: Phospho-proteomics, Transfection, Construct, Mutagenesis, Infection, Expressing, Western Blot, Incubation, Binding Assay, Immunoprecipitation