Pretrox tre3g retroviral plasmid

cDNA of CDCP1, siRNA-resistant CDCP1, Rab4a, Rab11a, ARHGEF7, shRNA-resistant ARHGEF7, and the PH domain of Btk were generated by PCR using human cDNA as the template and subcloned into the pCX4 retroviral plasmid (generously donated by Dr Akagi). siRNA-resistant CDCP1 and shRNA-resistant ARHGEF7 were generated by mutagenesis PCR. shRNAs against GEFs-mRNA were generated using PCR and a pLKO.1-shRNA vector and subsequently introduced by lentiviral infection. Detailed information on the primers is provided in Supporting information Table S1. All PCR experiments were performed using KOD-Plus polymerase (Toyobo Co, Ltd). Primer sequences used for mutagenesis and generation of shRNAs are listed in Supporting information Table S2. MET was subcloned into the pRetroX-TRE3G retroviral plasmid (Clontech Laboratories). All constructs were confirmed by sequencing. Gene transfer of pCX4 and pRrtroX-TRE3G was carried out by retroviral infection. Retroviral production was performed using Plat-E cells. Lipofection of viral vectors into Plat-E cells was performed using PEI MAX (Polysciences Inc). siRNAs were purchased from Sigma-Aldrich (St Louis) and transfected with Lipofectamine RNAiMAX (Thermo Fisher Scientific). siRNAs used are listed in Supporting information Table S3.

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)).