The ICSBP PCR product was cloned into the HindIII and XhoI sites of the pcDNA3.1/V5-HisA vector (Invitrogen, MA, USA). 143B and U2OS cells were transfected with the ICSBP construct or empty vector (Mock) using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Stable cell lines, including 143B-Mock and 143B-ICSBP cells, were established by selection with 500 μg/ml of geneticin (G418, Calbiochem, La Jolla, CA, USA) for 4 weeks.
Pcdna3.1 v5 hisa vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA3.1/V5-HisA vector is a plasmid DNA expression vector designed for the expression of recombinant proteins in mammalian cells. The vector contains a cytomegalovirus (CMV) promoter for high-level expression of the gene of interest, as well as a V5 epitope tag and a polyhistidine (6xHis) tag for the detection and purification of the expressed protein.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Cos 1, by American Type Culture Collection (1 mentions)
Cos 1, by Thermo Fisher Scientific (1 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Prl tk renilla, by Promega (1 mentions)
Pcmv lacz, by Takara Bio (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
5 fluorouracil 5 fu, by Merck Group (1 mentions)
Fugene 6 reagent, by Promega (1 mentions)
Pcdna3.1 myc his a vector, by Thermo Fisher Scientific (1 mentions)
Pegfp n1 vector, by Takara Bio (1 mentions)
9 protocols using pcdna3.1 v5 hisa vector
1
Stable Cell Line Generation with ICSBP Overexpression
2
Cell Line Culturing and Transfection
3
High-Throughput Nuclear Receptor Screening
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Promoters were tested for regulation by individual ligand-activated NR-members that heterodimerize with RXR (Table S1 ). For high-throughput screening, plasmids of NR/RXR heterodimers (15 ng NR + 15 ng RXR), promoter-reporters (30 ng) and internal controls (5 ng) pRL TK-Renilla (Promega, WI, USA) or pCMV-lacZ (Clontech CA, USA) were aliquoted in a volume of 10 μl/well into 384-well plates (Corning, 3707) using a 96-head liquid handling robot (Packard Evolution). The empty pcDNA3.1/V5-HisA vector (Invitrogen) was used to equalize the total amount of DNA transfected in all conditions. For reverse transfection, FuGENE HD (0.195 µl/well) and OptiMEM (4.805 µl/well) were mixed and 5 µl was added per well. After adding, the plate was gently shaken at RT and 35 µl of CV-1 cells (resuspended in phenol red-free DMEM medium with 10% charcoal-stripped serum) was added per well using a Titertek Multidrop 384 automated HT cell dispenser, making up a total volume of 50 µl per well. Plates were covered with a breathable seal and shaken gently before returning them to the incubator. The next day, 5 µl of ligand (Table S1 ) or control medium was added per well and incubated for another 24 h. The third day, plates were analyzed for luciferase activity using a luminometer (Wallac 1420 VICTOR2™ V, Perkin Elmer).
4
Modulating PLEKHA8P1 in FT3-7 cells
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FT3-7 cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), Penicillin-Streptomycin (Gibco, Waltham, MA, USA) in an incubator containing 5% CO2 at 37 °C. Cells were treated with 5-fluorouracil (5-FU) (Sigma, St. Louis, MO, USA) at indicated doses and time. Antisense oligonucleotides (ASOs) used to knockdown PLEKHA8P1 gene were designed and purchased alongside negative control ASOs from Qiagen. 5 × 105 cells were seeded in 6-well plates and ASOs (20 nM) were transfected into cells with Lipofectamine RNAiMax (Invitrogen, Waltham, MA, USA) as per manufacturer’s instructions. A second transfection was conducted after 24 h to increase knockdown efficiency. The ASO sequences are as follows:
Negative control ASO: 5′-AACACGTCTATACGC-3′,
PLEKHA8P1 ASO1: 5′-TTGCTGTGAAATCATG-3′,
PLEKHA8P1 ASO2: 5′-ACACTTTAGCACTTTA-3′.
For PLEKHA8 gene overexpression, PLEKHA8 sequence was cloned into pcDNA3.1 V5/His A vector (Invitrogen, Waltham, MA, USA). pcDNA3.1 PLEKHA8 V5/His A plasmid (4 μg, 6-well scale) was transfected into cells with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) as per manufacturer’s instructions.
Negative control ASO: 5′-AACACGTCTATACGC-3′,
PLEKHA8P1 ASO1: 5′-TTGCTGTGAAATCATG-3′,
PLEKHA8P1 ASO2: 5′-ACACTTTAGCACTTTA-3′.
For PLEKHA8 gene overexpression, PLEKHA8 sequence was cloned into pcDNA3.1 V5/His A vector (Invitrogen, Waltham, MA, USA). pcDNA3.1 PLEKHA8 V5/His A plasmid (4 μg, 6-well scale) was transfected into cells with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) as per manufacturer’s instructions.
5
Purification of His-tagged TME5
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TME5 was produced as previously described10 . Briefly, TME5 cDNA was amplified by PCR and was cloned into pcDNA3.1/V5-His-A vector (Invitrogen), followed by transfection into COS-1 cells. His-tagged TME5 were purified by using a His-tagged Protein PURIFICATION KIT (MBL, Nagoya, Japan).
6
Stable BEAS-2B Cytoglobin Overexpression
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The stable BEAS-2B over-expressing Cytoglobin (Cygb) by comprising the coding region of mouse Cygb which was cloned to pcDNA 3.1/V5-His A vector (Invitrogen) between Hind III and Xho I sites.
To establish stably Cygb expressing cells, a 1576 bp core ubiquitously-acting chromatin opening element (UCOE, a gift from Dr. Michael Antoniou, School of Medicine, King's College London, UK) was inserted to the upstream of the CMV promoter of pcDNA 3.1/V5-His A/Cygb. Successfully transfected cells were selected using selective medium (DMEM with 10% FBS and 600 µg/ml G418). After 14 days of selection, single colonies were picked and expression levels were checked to identify the clones with Cygb over-expression. The incorporation of the UCOE onto the Cygb–transgene construct resulted in the sustained high expression of cytogobin in the stable BEAS-2B cell line. This stable over-expression cell line was employed as a model system to investigate the effects of free radicals generated by ZEA on cell viability.
To establish stably Cygb expressing cells, a 1576 bp core ubiquitously-acting chromatin opening element (UCOE, a gift from Dr. Michael Antoniou, School of Medicine, King's College London, UK) was inserted to the upstream of the CMV promoter of pcDNA 3.1/V5-His A/Cygb. Successfully transfected cells were selected using selective medium (DMEM with 10% FBS and 600 µg/ml G418). After 14 days of selection, single colonies were picked and expression levels were checked to identify the clones with Cygb over-expression. The incorporation of the UCOE onto the Cygb–transgene construct resulted in the sustained high expression of cytogobin in the stable BEAS-2B cell line. This stable over-expression cell line was employed as a model system to investigate the effects of free radicals generated by ZEA on cell viability.
7
Seaweed Extracts Modulate Nuclear Receptors
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The LXR and PPAR-activating capacity of the seaweed extracts was determined in a cell-based reporter assay previously described by Zwarts et al. [59 (link)]. For this purpose, 1.0 × 106 cells were plated in T-25 culture flasks and after 24 h transfected by exposing the cells for 24 h to 1000 ng of pcDNA3.1/V5H6 vector containing clones of the full-length cDNAs for the murine nuclear receptors LXRα, LXRβ, PPARα or PPARγ, 1000 ng of vector encoding RXRα and 4000 ng of vectors encoding LXRE or PPRE using FuGENE® 6 reagent (Promega, Leiden, The Netherlands) according to the manufacturer’s instructions. Control conditions included cells transfected with 2000 ng of the RXRα-containing vector and 4000 ng of the LXRE- or PPRE-containing vector, and cells transfected with 2000 ng of an empty pcDNA3.1/V5-HisA vector (Invitrogen, Carlsbad, CA, USA) and 4000 ng of the LXRE- or PPRE-containing vector. All cells were co-transfected with 1000 ng of Renilla to normalize for variation in transfection efficiency.
8
Cloning and Mutagenesis of HDAC2
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HDAC2 expression vector was constructed by cloning the HDAC2 cDNA into a pcDNA 3.1/V5-His A vector (Invitrogen). HDAC2 sequence used in the rescue experiments was the following: ATCAACCCAGCGCTGTTGTTTTACAG. In addition, rescued-HDAC2 catalytic mutants expressing HDAC2A100 [35 (link)], HDAC2D142, HDAC2R11 were generated by using the QuickChange Lightning Site-Directed Mutagenesis kit (Stratagene) according to the manufacturers' protocol. Transfections of sh2 clones with rescued and HDAC2 mutants, were performed as previously described.
9
Cloning and Manipulation of Slitrk Proteins
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Full-length human Slitrk1 (aa 1–696) and human Slitrk2 (aa 1–845) were sub-cloned into the pEGFP-N1 vector (Clontech). For C-terminal V5- or MYC-tagged Slitrk1 constructs, full-length human Slitrk1 (aa 1–696) was sub-cloned into the pcDNA3.1 MYC-His A vector and the pcDNA3.1 V5-His A vector (Invitrogen), respectively. Slitrk1 mutants lacking either of the LRR domains (Slitrk1ΔLRR1 (Δ aa 18–264) and Slitrk1ΔLRR2 (Δ aa 304–599)) were also each sub-cloned into the pcDNA3.1 V5-His A vector. For knockdown of Slitrk1 in hippocampal neurons, shRNA sequences targeting nucleotides against rat Slitrk1 (Target #1: nucleotides 1222–1242; Target #2: nucleotides 2214–2234) were cloned into pcDNA6.2/GW-EmGFP-miR plasmid (gifted by Dr. Peter S. McPherson). PTPδ-Fc and HA-tagged Slitrk1 to Slitrk6 constructs were gifted to us by Dr. Hideto Takahashi and Dr. Ann Marie Craig, respectively.
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