Recombinant Protein Expression Strategies

cDNAs encoding proteins in this study were purchased from Addgene or synthesized by Integrated DNA Technologies. All mutations were generated by PCR and tagged with either FLAG or HA or His, and cloned into vectors by Gibson assembly. pCDNA3.1(+) vector (Invitrogen) was used for transient expression; viral vectors (Clontech) for stable expression; and pET-28a (Novagen) for bacterial expression.
Wild-type, BRAF^−/− and CRAF^−/− MEFs were generated in previous study [58 (link), 59 (link)]. Melanoma cell lines: MeWo, A101D, Mel-624 were obtained from ATCC.

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Yuan J., Ng W.H., Lam P.Y., Wang Y., Xia H., Yap J., Guan S.P., Lee A.S., Wang M., Baccarini M, & Hu J. (2018). The dimer-dependent catalytic activity of RAF family kinases is revealed through characterizing their oncogenic mutants. Oncogene, 37(43), 5719-5734.

Publication 2018

cDNAs pCDNA3.1(+) vector Mel-624

Bacterial Braf Cdnas Cell lines Craf Melanoma Mutations Proteins Transient Vectors

Corresponding Organization : Duke-NUS Medical School

Other organizations : National Cancer Centre Singapore, National University of Singapore, University of Vienna, Max Perutz Labs

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Variable analysis

independent variables

Mutations generated by PCR and tagged with either FLAG or HA or His
Cell lines: wild-type, BRAF−/− and CRAF−/− MEFs, MeWo, A101D, Mel-624

dependent variables

Unspecified, as no measured outcomes are explicitly mentioned

control variables

Vectors used for expression: pCDNA3.1(+) for transient expression, viral vectors (Clontech) for stable expression, and pET-28a (Novagen) for bacterial expression

controls

Positive control: Not specified
Negative control: Not specified

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