Pmscv mcherry syk

Manufactured by Addgene

The PMSCV-mCherry-SYK is a plasmid that contains the mCherry fluorescent protein fused to the SYK gene. The plasmid is designed for expression in mammalian cells.

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Lab products found in correlation

Aria fusion sorter, by BD (1 mentions) Quickchange lightning kit, by Agilent Technologies (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Pcmv5 shp2, by Addgene (1 mentions) Pcdna dest40 vector, by Thermo Fisher Scientific (1 mentions) Pcmv5 src, by Addgene (1 mentions) Pcgn ha n ras, by Addgene (1 mentions) Gateway cloning technology, by Thermo Fisher Scientific (1 mentions)

2 protocols using pmscv mcherry syk

Generation of SYK Knockout and Complemented HEK293T Cells

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HEK293T were transfected using FugeneHD transfection reagent (Promega) according to the provider’s manual with lentiCRISPRV2 plasmid containing the SYK gRNA sequence - CACACCACTACACCATCGAG. After 48 hr, bulk population was selected based on Puromycin sensitivity (1 μg/ml). Then, selected cells were seeded as single clones (1 cell / well) in 96-well plates. After 3-4 weeks, clones were screened for SYK expression by Immunoblot. HEK293T SYK KO line was complemented with mouse SYK cloned into pMSCV-mCherry-SYK (from Addgene, deposited by Hidde Ploegh). A SYK kinase-dead version (pMSCV-mCherry-SYK K396R) was generated in-house by QuickChange Lightning kit (Agilent Technologies) using the primers (gtgaaaaccgtggctgtgCGaatcctgaagaacgaggcc; ggcctcgttcttcaggattCGcacagccacggttttcac) and introduced into the HEK293T SYK CRISPR line using FugeneHD transfection reagent (Promega). After 48 hrs, bulk population was selected based on mCherry expression and cell sorted using an Aria Fusion Sorter (BD Biosciences).

Canton J., Blees H., Henry C.M., Buck M.D., Schulz O., Rogers N.C., Childs E., Zelenay S., Rhys H., Domart M.C., Collinson L., Alloatti A., Ellison C.J., Amigorena S., Papayannopoulos V., Thomas D.C., Randow F, & Reis e Sousa C. (2020). The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead cell-associated antigens. Nature immunology, 22(2), 140-153.

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Plasmid Construction and Characterization of Ras and Associated Proteins

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A plasmid encoding human pBabe-KRAS4B WT was generously provided by Channing Der (University of North Carolina, Chapel Hill), which was subcloned into pcDNA3 using KpnI and NotI to integrate an N-terminal HA tag. KRAS mutants (G12V, Y32F and Y64F) were generated by site-directed mutagenesis. Human pcDNA3-HA-HRAS, pCGN-HA-NRAS, pcDNA3-Flag-RAC2, pCDNA-HA-FAK P712/715A, pCDNA-HA-FAK Y576/577F, pMSCV-mCherry-SYK, pCMV5-Src (WT or K295RY527F), and pCMV5-SHP2 (WT, E76K or C459S) were obtained from Addgene. Flag-SHP2 constructs were subcloned into pcDNA3 and a plasmid encoding HA-CBL was subcloned into the pcDNA-DEST4.0 vector using Gateway Cloning technology (Invitrogen). Plasmids were verified by direct DNA sequencing.

Kano Y., Gebregiworgis T., Marshall C.B., Radulovich N., Poon B.P., St-Germain J., Cook J.D., Valencia-Sama I., Grant B.M., Herrera S.G., Miao J., Raught B., Irwin M.S., Lee J.E., Yeh J.J., Zhang Z.Y., Tsao M.S., Ikura M, & Ohh M. (2019). Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation. Nature Communications, 10, 224.

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