Pmcherry n1 plasmid

The BiFC assay was performed as described previously.⁶⁷ (link) To generate plasmids expressing split Venus fragments-tagged DDI2ΔUBL, the DDI2 cDNA was cloned into pBiFC-VN173 (Addgene_22010) or pBiFC-VC155 (Addgene_22011), respectively. Both BiFC plasmids were co-transfected with a pmCherry-N1 plasmid (Clontech), which was used as an internal control. At one day after transfection, the cells were cultured without all amino acids for 3 h. The fluorescent intensities of Venus and mCherry were measured using a flow cytometer (FACSAriaII, BD Biosciences). Median fluorescence intensity (MFI) values of Venus in mCherry-positive cells were measured by flow cytometry.

CDCP1, CDCP1 deletion mutants, STAT3, and Met were generated by PCR using human cDNA as the template and subcloned into the pCX4 retroviral plasmid (generously donated by Dr. Akagi) (76 (link)). CDCP1 mutants (K365A-R368A-K369A, C689G-C690G, Y762F, and Y734F) and STAT3-Y705F were generated by mutagenesis PCR using KOD-Plus polymerase (Toyobo). CDCP1 and its respective mutants were subcloned into either pEGFP-N1 or pmCherry-N1 plasmid (Clontech) and then further subcloned into the pRetroX-TRE3G retroviral plasmid (Clontech). Src-MER and STAT3-MER were constructed by modifying ligand-binding domain of the estrogen receptor (MER, amino acids 281–599) and subcloning into the pCX4 plasmid (27 (link)). mCherry-CAAX was constructed using the C-terminal region of human KRAS (amino acids 166–189) and subcloning into the pCX4 plasmid. mCherry-GPI was also subcloned into the pCX4 plasmid (generously donated by Dr. Kiyokawa) (77 (link)). All constructs were confirmed by sequencing. Gene transfer of pCX4 and pRertroX-TRE3G was carried out by retroviral infection. Retroviral production and infection were performed as described previously (78 (link)).

pCMV-EGFP retrovirus (Betizeau et al., 2013 (link)) were produced by M. Afanassieff (SBRI, INSERM U1208 Bron, France) via pTG5349, pTG13077 (Transgene SA, Illkirch-Graffenstaden, France), and phCMV-G [Gift from D. Nègre ENS Lyon (Yee et al., 1994 (link))].
mCherry construct is as follows. NheI-mcherry-XhoI PCR of mCherry cDNA, from pmCherry-N1 plasmid from Clontech (PT3974-5), was cloned into modified pEGFPC1 plasmid from Clontech (ref 6084-1) where EGFP-C1 was previously switched for MCS NheI-SmaI-EcoRV-ClaI-XhoI.