Pcmv5 shp2

Manufactured by Addgene

PCMV5-SHP2 is a plasmid vector that expresses the SHP2 protein. SHP2 is a protein tyrosine phosphatase that plays a role in various cellular signaling pathways.

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Lab products found in correlation

Pcgn ha n ras, by Addgene (2 mentions) Pcmv5 src, by Addgene (2 mentions) Gateway cloning technology, by Thermo Fisher Scientific (2 mentions) Pmscv mcherry syk, by Addgene (1 mentions) Pcdna dest40 vector, by Thermo Fisher Scientific (1 mentions)

3 protocols using pcmv5 shp2

Plasmid Constructs for Ras, Src, and SHP2

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Plasmids encoding human pCGN-HA-N-Ras(WT or 12D), pCMV5-c-Src(WT or K295R) and pCMV5-SHP2(WT) were obtained from Addgene. Flag-SHP2 constructs were obtained from Addgene and subcloned into pcDNA3. Plasmids were verified by direct DNA sequencing. Monobodies directed against SHP2 were generated as previously described24 (link) and generously provided by Dr Shohei Koide.

Bunda S., Burrell K., Heir P., Zeng L., Alamsahebpour A., Kano Y., Raught B., Zhang Z.Y., Zadeh G, & Ohh M. (2015). Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis. Nature Communications, 6, 8859.

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Cloning and Mutagenesis of KRAS Variants

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A plasmid cDNA encoding human KRAS4B WT was subcloned from a pBabe plasmid (generously provided by Dr. Channing Der, University of North Carolina, Chapel Hill) into pcDNA3. KpnI and NotI restriction enzymes were used to integrate an N-terminal HA tag to the plasmid. We conducted site-directed mutagenesis (Quikchange; Roche) to generate single and double KRAS mutants (G12V, G12D, Q61H, Y32F/Q61H, and Q61H/Y64F). pCMV5-Src (WT or K295RY527F), pCMV5-SHP2 (WT and C459S), pCGN-HA-HRAS WT, pCGN-HA-KRAS WT, and pCGN-HA-NRAS WT were obtained from Addgene. Gateway Cloning technology (Invitrogen) was used to subclone Flag-SHP2 constructs into pcDNA3. All the plasmids were verified by direct DNA sequencing before they were used in this study.

Gebregiworgis T., Kano Y., St-Germain J., Radulovich N., Udaskin M.L., Mentes A., Huang R., Poon B.P., He W., Valencia-Sama I., Robinson C.M., Huestis M., Miao J., Yeh J.J., Zhang Z.Y., Irwin M.S., Lee J.E., Tsao M.S., Raught B., Marshall C.B., Ohh M, & Ikura M. (2021). The Q61H mutation decouples KRAS from upstream regulation and renders cancer cells resistant to SHP2 inhibitors. Nature Communications, 12, 6274.

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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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