U2OS human osteosarcoma, HEK-293 human embryonic kidney, and NCI-H460 human non-small cell lung carcinoma cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (U2OS and HEK-293) or RPMI (NCI-H460) medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in 5% CO2. The pMLM3636 expression vector for Streptococcus pyogenes Cas9 sgRNA was a gift from JK Joung laboratory (Addgene plasmid # 43860). The Cas9D10A expression vector was a gift from G Church (Addgene plasmid #41816) (19 (link)), the CAGA12-Luc plasmid was described in (20 (link)), and the Renilla expression vector pRL-TK vector is from Promega. The pCMV10-3xFlag-RNF111-WT (Flag-RNF111-WT) expression vector was generated by PCR subcloning of human RNF111 cDNA (corresponding to isoform 3) from PcDNA4/TO-SFS-RNF111 (11 (link)) in pCMV10-3xFlag (Sigma). The pCMV-3xHA-SKIL-WT (HA-SKIL-WT) expression vector was generated by PCR subcloning of human SKIL cDNA form PCMV5B-HA-SnoN (21 (link)) in PCMV-3xHA. pCMV10-3xFlag-RNF111-C933A (Flag-RNF111-C933A) and pCMV-3xHA-SKIL-342/43-KR (HA-SKIL-342/43-KR) mutants were generated by site-directed mutagenesis respectively on pCMV10-3xFlag-RNF111-WT and pCMV-3xHA-SKIL-WT by using the QuickChange Lightning kit (Agilent).
Pcmv10 3x flag
Manufactured by Merck Group
Sourced in United States
PCMV10-3x-FLAG is a laboratory equipment product developed by Merck Group. It is a mammalian expression vector that contains a cytomegalovirus (CMV) promoter and a 3x-FLAG tag sequence. The core function of this product is to facilitate the expression and purification of recombinant proteins in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Polyethyleneimine reagent, by Merck Group (1 mentions)
Pcdna3.1 myc his vector, by Thermo Fisher Scientific (1 mentions)
Polyethyleneimine, by Merck Group (1 mentions)
Cdna synthesis kit, by Toyobo (1 mentions)
Prl tk vector, by Promega (1 mentions)
Quickchange lightning kit, by Agilent Technologies (1 mentions)
Pmlm3636, by Addgene (1 mentions)
3 protocols using pcmv10 3x flag
1
Generating Engineered Cell Lines for CRISPR Research
2
Mutational Analysis of NS5A Phosphorylation
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Myc-tagged NS5A and GFP-tagged NS5A were described previously (Nguyen et al., 2020 (link)). Substitutions of serine to alanine at 2194, 2197, 2201, and 2204 hyperphosphorylation sites in NS5A were performed using a QuikChange® II XL Site-Directed Mutagenesis Kit (Stratagene, USA) according to the manufacturer’s instructions. IFN-β-luc and ISRE-luc plasmids were provided by Dr. S. Goodbourn (St George’s, University of London, UK). Flag-tagged IKKε wild-type, K38A, S172A, and TBK1 plasmids were provided by Dr. Kate Fitzgerald (University of Massachusetts, USA). Flag-tagged IKKε mutants (1-383, 1-299, 384-717, and 300-717) were generated by polymerase chain reaction (PCR) amplification of the relevant sequences from Flag-tagged wild-type IKKε and inserted into pCMV10-3x-FLAG (Sigma-Aldrich, USA). Myc-tagged and His-tagged DDX3 plasmids were provided by Dr. Andrew G. Bowie (Trinity College Dublin, Ireland). pEF-BOS-Flag-RIG-I, pEF-BOS-Flag-MDA5, and pEF-BOS-Flag-MAVS were provided by Dr. Takashi Fujita (Kyoto University, Japan). Full-length IRF3 was amplified by PCR using cDNA prepared from HEK293T cells and was subcloned into the pcDNA3.1/Myc-His vector (Invitrogen, USA). All DNA transfections were performed using a polyethyleneimine reagent (Sigma-Aldrich) as we described previously (Nguyen et al., 2020 (link)).
3
Cloning and Expression of ANKRD1 and HCV Proteins
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Full-length ANKRD1 was amplified by primers (Supplementary Table 2 ) from cDNA synthesized from Huh7.5 cells by using cDNA synthesis kit (TOYOBO) according to manufacturer’s instructions. PCR products were inserted into the BglII/BamHI sites of the plasmid pCMV10-3x Flag (Sigma Aldrich). ANKRD1 mutants were generated by PCR and subcloned into pCMV10-3x Flag vector. Plasmids expressing NS5A mutants, Myc-tagged HCV core, NS3, NS4B, NS5A, NS5B, and GST-NS5A were described elsewhere42 (link). All DNA transfections were performed by using polyethyleneimine (Sigma-Aldrich) as we described previously42 (link).
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