Pcdh cmv mcs ef1 puro lentivector

shRNAs pLKO.1 lentiviral plasmids used for non-targeting shRNA control (SHC002) and human PRKDC knockdown were purchased from Sigma. The shRNA clones used for targeting PRKDC were TRCN0000197152 (shPRKDC#1), TRNC0000196328 (shPRKDC#2), TRCN0000195491 (shPRKDC#3), TRCN0000194985 (shPRKDC#4) and TRCN0000194719 (shPRKDC#5). Non-targeting control shRNA and three PRKDC shRNAs (shPRKDC#1, shPRKDC#3 and shPRKDC#5) were cloned into the inducible pLKO-Tet-On puromycin vector as previously described [26 (link)]. pCDH-CMV-MCS-EF1-Puro lentivector was purchased from System Bioscience. pCDH-c-MYC, pCDH-L-MYC1, pCDH-L-MYC2 and pCDH-N-MYC vectors were generated by cloning protein coding sequences of human c-MYC, L-MYC isoform1, L-MYC isoform2 and N-MYC into pCDH-CMV-MCS-EF1-Puro lentivector. Etoposide, NU-7441, and KU0060648 were obtained from Sigma (cat# E1383), Tocris Cookson Inc. (cat# 3712), and Axon Medchem BV (cat# Axon 1584), respectively. The proteasome inhibitor, MG132, was obtained from Sigma (cat#C2211).

The cDNA encoding rat NPC1L1 (rNPC1L1) (GenBank AY437867) was generated by PCR amplification from rat jejunum single-strand cDNA (GenoStaff) and cloned into pCR4 Blunt TOPO (Life Technologies). The rNPC1L1 cDNA from this construct was inserted into the expression vector pcDNA3.1 (Life Technologies) and pCDH-CMV-MCS-EF1-Puro Lentivector (System Biosciences).
The cDNA encoding the human NPC1L1 (hNPC1L1) (GenBank AY437865) was generated by PCR amplification from a human liver cDNA library (Takara Bio) and cloned into pCR2.1 (Life Technologies). The hNPC1L1 cDNA was modified by inserting enhanced green fluorescent protein (EGFP) sequence at the C terminus, and then ligated into pcDNA3.1. Site-directed mutagenesis was performed by a PCR-based strategy using iProof high-fidelity DNA polymerase (Bio-Rad Laboratories). The influenza virus hemagglutinin (HA) tag was inserted between S986 and L987 of hNPC1L1.
The pcDNA3.1 constructs encoding rNPC1L1, hNPC1L1-EGFP, hNPC1L1^{L1072T/L1168I}-EGFP, HA-hNPC1L1-EGFP, or HA-hNPC1L1^{L1072T/L1168I}-EGFP were transfected into HEK293 cells using FuGENE HD (Promega). Cells were selected by culturing in the presence of 1 mg/ml G418 sulfate (Wako Pure Chemical Industries). For FACS analysis, HEK293 cells stably expressing HA-hNPC1L1^{L1072T/L1168I}-EGFP were cloned by limiting dilution.