The, pL-Oct-1R-3FLAG (C-end) construct was generated by inserting a copy of the human Oct-1R coding sequence into the pLenti6/V5-D-TOPO expression vector (Invitrogen).
Plenti6 v5 d topo expression vector
Manufactured by Thermo Fisher Scientific
The PLenti6/V5-D-TOPO expression vector is a lentiviral-based system designed for high-level, constitutive expression of recombinant proteins in a wide range of mammalian cell types. The vector includes the V5 epitope tag for detection and purification of the expressed protein, as well as the blasticidin resistance gene for selection of stable cell lines.
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4 protocols using plenti6 v5 d topo expression vector
1
Generation of pL-Oct-1R-3FLAG Construct
2
Lentiviral Transduction of FADD-DN
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The sequence of FADD-DN was cloned into a plenti6/v5-D-Topo expression vector (Invitrogen) using restriction enzyme BamHI and XhoI. Primers for amplifying the DNA sequence of FADD-DN: forward: 5′-CGGGATCCATGGACGACTTCGA-3′ and reverse: 5′-CCCTCGAGTCAGGACGCTTCGGAGGT-3′. Briefly, HEK-293T cells were co-tranfected with FADD-DN expression plasmid and lentiviral envelope plasmids (PL3, PL4, and PL5). The viruses were harvested by ultra-centrifugation on day 3 after transfection.
Jurkat T cells was replaced with transduction medium. 8 μg/ml polybrene (Santa Cruz Biotechnology) was added followed by lentiviral transduction. Virus was removed 24 h after transduction and 10 μg/ml blasticidin was added for another 48 h. The cells were cultured for 72 h to examine the expression of FADD-DN by western blot.
Jurkat T cells was replaced with transduction medium. 8 μg/ml polybrene (Santa Cruz Biotechnology) was added followed by lentiviral transduction. Virus was removed 24 h after transduction and 10 μg/ml blasticidin was added for another 48 h. The cells were cultured for 72 h to examine the expression of FADD-DN by western blot.
3
Generation of Oct-1 Lentiviral Constructs
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The constructs, pL-Oct-1A-3FLAG, pL-Oct-1L-3FLAG, pL-Oct-1X-3FLAG (C-end) were generated by inserting a copy of human Oct-1 coding sequences into the pLenti6/V5-D-TOPO expression vector (Invitrogen).
4
Lentiviral Transduction of PIM2 and RIPK2 in HCC
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Full-length of human PIM2 gene was PCR amplified and cloned into pLenti6/v5-D-topo expression vector (Invitrogen) according to manufacturer’s instructions. PIM2 containing lenti-virus was then stably transduced into HCC cell lines by blasticidin selection. Empty vector transduced cells were used as controls. Two short hairpin RNAs (shRNA) specifically targeting on PIM2 or specifically targeting on RIPK2 were cloned into pLL3.7 lenti-viral vector. HCC cell lines were transduced with shRNAs to establish stable knockdown cell lines. See the Supplementary Materials and Methods section for details.
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