To determine whether the deletion of the Hsd17b1 gene affects Naglu expression in vitro, we generated two expression constructs in pACYC177 plasmid backbone (New England Biolabs, Ipswich, MA, USA) of this genomic region with and without the genomic fragment spanning the 1.8 kb coding region of Hsd17b1 (Fig. 1F and G ). The constructs were co-transfected with a luciferase-expressing reporter plasmid (pRL Renilla luciferase reporter vector, Promega, Madison, WI, USA) into COS cells using TurboFect kit (Thermo Fisher Scientific, Wilmington, DE, USA) according to the instructions supplied by the manufacturer. Thereafter, the expression of Naglu in the transfected cells was analysed by qRT-PCR and normalised to luciferase expression (n = 6).
Turbofect kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TurboFect kit is a transfection reagent designed for efficient and convenient delivery of nucleic acids, such as plasmid DNA, into a variety of cell lines. The kit provides a simple and effective method for introducing genetic material into cells, facilitating various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Prl renilla luciferase reporter vector, by Promega (1 mentions)
Erk1 2 9102, by Cell Signaling Technology (1 mentions)
Akt 9272, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
G9545, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Kod plus neo kit, by Toyobo (1 mentions)
5 protocols using turbofect kit
1
Hsd17b1 Deletion and Naglu Expression
2
Western Blot Analysis of Rac1, ERK, and AKT
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SDS-PAGE (10%) was performed and Rac1 was detected using a rabbit polyclonal antibody #2465 (Cell Signaling Technology, Leiden, The Netherlands). GAPDH was used as a loading control #G9545 (Sigma-Aldrich, Taufkirchen, Germany). For analysis of ERK and AKT phosphorylation, cells were transfected using the TurboFect kit (Thermo Fisher Scientific, Rockford, Illinois, USA), left for 2 days in medium containing fibronectin-depleted FCS and harvested. The antibodies used were: pERK 1/2 (detecting phosphorylation at Thr202/Tyr185) #4376, ERK 1/2 #9102, pAKT (detecting phosphorylation at Ser473) #9271, AKT #9272 (All four antibodies from Cell Signaling Technology, Leiden, The Netherlands).
3
Biliverdin Enrichment of Transfected Cells
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HeLa (ATCC CCl-2) and U2OS (ATCC HTB-96) cells were transfected with the respective plasmid using TurboFect Kit (Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM-Medium: 4.5 g/L glucose, GlutaMAX, phenol red, 10% vol/vol FCS, 1 mM sodium pyruvate, 100 µg/ml streptomycin, 100 µg/ml penicillin) on coverslips (for imaging) or without coverslips (for FACS measurements) in 6-well plates at 37 °C, 90% humidity and 5% CO2. Approximately, 2 h before imaging or measuring, 25 µM biliverdin was added to the medium.
4
Cloning and Validating TUB Gene Variants
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The 2240-bp DNA fragments of TUB (including exons 10–12 and introns 10–11) were amplified from the genomic DNA of the proband or a healthy control with primers as the following: TUB-BamHI-F: 5′- aagcttggtaccgagctcggatccGTCCAACTTGATGGGCACCAAGTTCACT-3′; TUB-XhoI-R: 5′- ttaaacgggccctctagactcgagGGTCATTGCCATGGATGATCTGGAAGTT-3′. After treatment with restriction endonucleases BamHI and XhoI, the amplified fragments were cloned into pMini-CopGFP using the KOD-Plus Neo Kit (TOYOBO CO., OSAKA, JAPAN). The recombinant constructs were confirmed by Sanger sequencing. The wild type (pMini-CopGFP-TUB-WT) and mutant type (pMini-CopGFP-TUB-MT) recombinant constructs were then transiently transfected into two commonly used mammalian cell lines (APRE-19 and 293 T cells) following the protocol provided by the TurboFect kit (Thermo, USA). RT-PCR was performed to obtain the target fragments of human TUB gene with primers as the following: MiniRT-F: 5’-GGCTAACTAGAGAACCCACTGCTTA-3’; TUB-RT-R: 5’-GGTCATTGCCATGGATGATCTG-3’. The amplified fragments were detected on 1.5% agarose gel and further verified by Sanger sequencing.
5
Expression of NS3 Proteins in Transfected Cells
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Expression of NS3 fragment was analyzed in transfected 293T cells with the pc-NS3 by the turbofect kit (Thermo Scientific, CA). One day after transfection, the medium was changed with fresh medium to grow for 48 hours. Finally, the transfected cells were collected to detect the NS3 proteins. The un-transfected cell served as the negative control. The collected cells were washed twice in phosphate buffered saline (PBS) and treated with lysis buffer (1% Nonidet P-40, 10 mg/L- phenyl methylsulfonyl fluoride 50 mM-tris Cl, pH 8.0) for 30 minutes on ice and then the lysates were centrifuged at 800 g for 15 minutes. The supernatants were collected to be analyzed by the western-blot method.
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