C-terminal HA-tagged TRIM5αhu and TRIM5αrh were obtained by PCR using cDNA from HEK293 and RhFK-4 cells respectively. Then stably overexpressing cell lines were constructed as previously described [18 (link)]. For the TRIM11 stable knockdown cell lines, the shRNA were constructed into pLKO.1 (Ctrl: TTCTCCGAACGTGTCACGT, 1:TATTCATCTTTCCCGAGAT, 2:CTATTACAATTCCTCGGAA). HEK293T cells were co-transfected with pLKO.1, psPAX2 and pMD2.G using Fugene HD Transfection Regent (Promega). Lentiviruses were collected at 48 h after transfection, filtered through 0.45 μm filters. HEK293 cells or THP-1 cells were infected with the lentivirus, and 24 h later infected cells were selected in medium containing 1.1 μg/ml puromycin. For THP-1 cells, the selective concentration of puromycin was 2.7 μg/ml.
Fugene hd transfection regent
Manufactured by Promega
Sourced in Japan, United States
FuGENE HD Transfection Reagent is a non-liposomal, multi-component, proprietary formulation that facilitates the delivery of DNA into a variety of mammalian cell types. It is designed to provide high transfection efficiency with low cytotoxicity.
Automatically generated - may contain errors
Lab products found in correlation
Primescript 2 reverse transcriptase, by Takara Bio (1 mentions)
Tks gflex dna polymerase, by Takara Bio (1 mentions)
Kod plus mutagenesis kit, by Toyobo (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Primestar mutagenesis basal kit, by Takara Bio (1 mentions)
Prism 5, by GraphPad (1 mentions)
Plenti cmv puro luc w168 1, by Addgene (1 mentions)
Steady glo luciferase assay system, by Promega (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Pgl4.23 firefly luciferase vector, by Promega (1 mentions)
7 protocols using fugene hd transfection regent
1
Stable Overexpression and Knockdown of TRIM Proteins
2
Establishment of Inducible Tbx6 Expressing mESCs
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Total RNA was extracted from ICR 10.5 dpc mouse embryos and converted to cDNA using PrimeScript II reverse transcriptase (Takara-bio). The coding sequence of mouse Tbx6 gene was isolated by PCR using Tks Gflex DNA polymerase (Takara-bio) from this cDNA. After confirmation of no mutation by DNA sequencing, it was subcloned into pPthc vector. EBRTcH3 mESCs were obtained from Riken Bioresource Center (Tsukuba, Japan), and were co-transfected with pPthc-Tbx6 and pCAGGS-Cre under the presence of dox using Fugene HD transfection regent (Promega), and subjected to selection with puromycin (FOCUS Biomolecules) [12] (link). The expected structure of rosa-Tet locus of puromycin-selected cells is shown in Fig. 1 A. The resultant cell line, EBRTcPTbx6, was used in this study.![]()
Establishment of EBRTcPTbx6 cells. (A) Construct of Rosa26 locus in EBRTcPTbx6 cells. (B–C) Response of EBRTcPTbx6 cells to dox. In dox+ conditions, cells showed no fluorescence (B) while in dox− conditions cells were green fluorescent (C). (D–E) Immunostaining with anti-Oct-3/4 antibody showed positive signals in EBRTcPTbx6 cell nuclei under dox+ conditions (D), with merged image with DAPI counterstain (E), (F) RT-PCR of Tbx6-venus and GAPDH genes in EBRTcPTbx6 cells. Tbx6-venus transcript was only detected under dox− conditions.
3
Expression and Mutagenesis of SIK Kinases
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HEK293 cells (RCB2202) were obtained from the RIKEN BRC Cell Bank. HEK293 cells were cultured and passaged under 5% CO2 in DMEM containing 10% fetal bovine serum and penicillin/streptomycin. Then, 3xFLAG-tagged mouse Sik1, mouse Sik2, and mouse Sik3 cDNAs were cloned into the pcDNA3.1 vector using an In-Fusion HD Cloning Kit (TaKaRa). Although there are multiple protein isoforms of SIK3, we found that SIK3 isoform ending at exon 14 (e.g. UniProt: Q6P4S6-2) is a major form (Ikkyu et al., unpublished data). We used this SIk3 isoform in this study. Expression vectors for FLAG-SIK1 (S577A) and FLAG-SIK2 (S587A) were generated from pcDNA3-FLAG-SIK1 and pcDNA3-FLAG-SIK2 using a KOD-Plus-Mutagenesis Kit (Toyobo) and a PrimeSTAR Mutagenesis Basal Kit (TaKaRa). Cells were grown to 80% confluency in 6-well plates. HEK293 cells were transfected with 3 μg of pcDNA for FLAG-SIK1, FLAG-SIK1 S577A, FLAG-SIK2, FLAG-SIK2 S587A, 3xFLAG-SIK3, and 3xFLAG-SIK3 S551A by using 9 μl of FuGENE HD Transfection Regent (Promega).
4
Tagging Jam1a and Jam2a proteins
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A Flag and HA tag were inserted between the two Ig domains of jam1a and jam2a by two-step PCR, respectively, and cloned into the pcDNA3 vector as previously described29 (link). HEK293T or Hela cells were transiently transfected with the above constructs using FuGENE HD transfection regent (Promega, E2311). Cells were then cultured in 6-well plate with 10%FBS, 200mM L-glutamine in Dulbecco's Modified Eagle Medium (DMEM) at 37°C and 5% CO2.
5
Lentiviral Vector Transduction Assay
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To generate the luciferase reporter lentiviral vector, 293T cells (4 × 106 cells in a 100 mm dish) were transfected with pCMV‐ΔR8.91 or the derivative, a lentiviral vector plasmid, pLenti CMV Puro LUC (w168‐1) (Addgene, Cambridge, MA, USA) 23 and a vesicular stomatitis virus G protein expression plasmid, pHit/G 24 using FuGENE HD Transfection Regent (Promega, Madison, WI, USA). Seventeen hours after transfection, 293T cells were transferred into 96‐well plate and serially diluted PIs were added in each well. Thirty‐one hours later, 293T cells, seeded as target cells 24 h prior to the infection in 96‐well plate, were infected with lentiviral vector in the culture supernatant of plasmid‐transfected cells. Twenty‐four hours after infection, luciferase activity in infected cells was measured using a Steady‐Glo Luciferase Assay System (Promega) with an LB962 microplate luminometer (Berthold, Bad Wildbad, Germany). Drug susceptibility was evaluated as a reduction in luciferase activity in infected cells. The 50% inhibitory concentration (IC50) of PIs for suppressing the transduction of luciferase gene was calculated from the dose–response curve using a standard function of graphpad prism 5 software (GraphPad Software, San Diego, CA, USA).
6
Tagging Jam1a and Jam2a proteins
7
Sp9 Gene Enhancer Luciferase Assay
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