Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN (Fiordalisi et al., 2001 (link)). Amino acid substitutions were generated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis kit (Agilent Technologies). All plasmids were verified by bidirectional sequencing.
For siRNA knockdown, cells were transfected with ON-TARGETplus SMARTpools (GE Healthcare) targeting VPS35, PDE6D or nontargeting control using DharmaFECT 1 reagent according to the manufacturer’s instructions. For shRNA knockdown, targeting sequences for VPS35 were inserted into pTRIPz lentiviral vector by PCR, and lentiviral particles were generated with standard protocols (http://tcf.epfl.ch/files/content/sites/tcf/files/shared/LV_production.pdf ). pTRIPz-based NRAS-targeting lentivirus vectors (Grabocka et al., 2014 (link)) were provided by D. Bar-Sagi (New York University Langone Medical Center, New York, NY). Stable cells lines were generated by lentiviral infection and selection with puromycin for 5 d. Knockdown was induced by 0.4 µg/ml doxycycline for at least 72 h.
For siRNA knockdown, cells were transfected with ON-TARGETplus SMARTpools (GE Healthcare) targeting VPS35, PDE6D or nontargeting control using DharmaFECT 1 reagent according to the manufacturer’s instructions. For shRNA knockdown, targeting sequences for VPS35 were inserted into pTRIPz lentiviral vector by PCR, and lentiviral particles were generated with standard protocols (
