Pdest mcherry n1

Manufactured by Addgene

Sourced in United States

The PDEST-mcherry-N1 is a plasmid that enables the expression of mCherry, a red fluorescent protein, in mammalian cells. This plasmid can be used for various applications that require the visualization of protein localization or expression patterns.

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Lab products found in correlation

Pcdna6.2 c emgfp dest, by Thermo Fisher Scientific (1 mentions) Pcdna6.2 c yfp dest, by Thermo Fisher Scientific (1 mentions) Pcdna6.2 v5 dest, by Thermo Fisher Scientific (1 mentions) Ripa buffer, by Merck Group (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)

2 protocols using pdest mcherry n1

GM-CSF Receptor Subunits Interaction

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The complete coding regions of CSF2RA, CSF2RB_WT and CSF2RB_Mut were synthesized by GeneArt gene synthesis and cloned into the Gateway pDONR™221 Vector (GeneArt, Life Technologies), and the clone sequences confirmed by Sanger sequencing. For immunoblot analyses, CSF2RB_WT and Mut were sub-cloned into the Gateway destination vector pcDNA6.2_C-EmGFP-DEST (Life-Technologies), and CSF2RA into pcDNA6.2_C-YFP-DEST (Life-Technologies). For co-localization studies, CSF2RA and CSF2RB_WT were sub-cloned into PCDNA6.2/V5-DEST (Life-technologies) and pDEST-mcherry-N1 (Addgene plasmid # 31907), respectively.
HEK293 cells (ATCC CRL 1573) were seeded at 2×10⁵ cells per well in 24 well plates and transfected with 500ng of total DNA with Lipofectamine 3000 (Life Technologies). Transfected DNA was comprised of combinations of pcDNA6.2_C-YFP-DEST -CSF2RA, pcDNA6.2_C-EmGFP-DEST-CSF2RB_WT, and pcDNA6.2_C-EmGFP-DEST-CSF2RB_Mut (Life Technologies). Twenty-four hours post-transfection, cells were stimulated with 0, 0.4, 2 or 10 ng/ml of GM-CSF for 15 min and lysed in RIPA buffer (Millipore). For co-localization studies, CSF2RA, CSF2RB_WT and CSF2RB_Mut were co-transfected and stimulated with GM-CSF (10 ng/ml).

Chuang L.S., Villaverde N., Hui K.Y., Mortha A., Rahman A., Levine A.P., Haritunians T., Evelyn Ng S.M., Zhang W., Hsu N.Y., Facey J.A., Luong T., Fernandez-Hernandez H., Li D., Rivas M., Schiff E.R., Gusev A., Schumm L.P., Bowen B.M., Sharma Y., Ning K., Remark R., Gnjatic S., Legnani P., George J., Sands B.E., Stempak J.M., Datta L.W., Lipka S., Katz S., Cheifetz A.S., Barzilai N., Pontikos N., Abraham C., Dubinsky M.J., Targan S., Taylor K., Rotter J.I., Scherl E.J., Desnick R.J., Abreu M.T., Zhao H., Atzmon G., Pe’er I., Kugathasan S., Hakonarson H., McCauley J.L., Lencz T., Darvasi A., Plagnol V., Silverberg M.S., Muise A.M., Brant S.R., Daly M.J., Segal A.W., Duerr R.H., Merad M., McGovern D.P., Peter I, & Cho J.H. (2016). A Frameshift in CSF2RB Predominant Among Ashkenazi Jews Increases Risk for Crohn’s Disease and Reduces Monocyte Signaling via GMCSF. Gastroenterology, 151(4), 710-723.e2.

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Fluorescent Fusion Protein Generation

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Wild type FAK and FAK Y397F were amplified by PCR from pAcGFP1-Hyg-C1-FAK wild type and Y397F (a kind gift from P. Rondé, LBP, Strasbourg) with appropriate primers (Table S2). PCR products were used to generate pDONR-FAK and pDONR-FAK Y397F, respectively, before subcloning in the destination vector pDEST-mCherry C1 (a gift from F. Lembo, CRCM) by Gateway technology, to obtain FAK and FAK Y397F fused to the C-terminus of mCherry. Wild type EB1 was amplified by PCR from peGFP-N1-EB1 (#39299; Addgene, a gift from T. Mitchison and J. Tirnauer, Harvard) with appropriate primers (Table S2). PCR products were used to generate pDONR-EB1 before subcloning in the destination vector pDEST-mCherry N1 (#31907, Addgene, a gift from R.M. Shaw, San Francisco, CA, USA). All constructs were sequence verified.

Chanez B., Ostacolo K., Badache A, & Thuault S. (2021). EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK. Cells, 10(2), 388.

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