The expression plasmid encoding the FLAG-tagged Acin1-L isoform (pCAGGS-Acin1-L) was a kind gift provided by Dr. Christian Schwerk (University of Mahhheim). The open reading frame (ORF) of the human Acin1-S isoform was amplified with a polymerase chain reaction (PCR) using a human fetal cDNA library as the template. The PCR product digested with Hind III/BamH I was inserted into the p3XFLAG-CMV14 vector (Sigma, St. Louis, MO, USA). ORFs of human MBNL1 isoforms 2 and 8 were amplified with a PCR using a human fetal cDNA library as the template. The PCR product digested with Kpn I/BamH I was inserted into the p3XFLAG-CMV14 vector (Sigma). The human genomic element containing the cassette exons of MBNL1 was PCR-amplified using human genomic DNA as the template. The PCR product was digested with Kpn I and BamH I restriction enzymes and inserted into pcDNA 3.1 vector (Invitrogen). Derived mutants of the splicing reporter or expressing vector containing the substituted nucleotide were constructed using the QuikChange site-directed mutagenesis system (Stratagene, Amsterdam, The Netherlands). Vector-based short hairpin (sh)RNAs targeting the human MBNL1 or Acin1 gene were purchased from the RNAi core facility at Academia Sinica (Taipei, Taiwan).
P3xflag cmv14 vector
Manufactured by Merck Group
Sourced in United States
The P3XFLAG-CMV14 vector is a laboratory tool used for recombinant protein expression. It is designed to enable the production of proteins with a 3XFLAG tag in mammalian cell lines. The vector contains a cytomegalovirus (CMV) promoter to drive high-level transgene expression.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Mission sirna, by Merck Group (1 mentions)
293ltv cell line, by Cell Biolabs (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Doxycycline hyclate, by Merck Group (1 mentions)
Pmdlg prre, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Prl tk vector, by Promega (1 mentions)
Nepa21 electroporator, by Nepa Gene (1 mentions)
Pretrox tre3g vector, by Takara Bio (1 mentions)
His ub, by Addgene (1 mentions)
Puc19 vector, by New England Biolabs (1 mentions)
Dna ligation kit, by Takara Bio (1 mentions)
Bamhi, by Takara Bio (1 mentions)
Anti flag m2 agarose, by Merck Group (1 mentions)
Prl sv40 vector, by Promega (1 mentions)
Pegfp n3 vector, by Takara Bio (1 mentions)
Pflag cmv2 vector, by Merck Group (1 mentions)
18 protocols using p3xflag cmv14 vector
1
Molecular Cloning of MBNL1 and Acin1 Isoforms
2
Cloning and Characterization of β-TrCP Proteins
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The cDNAs of β-TrCP2 was cloned in mammalian expression vector pCMV6-Entry having C-terminal FLAG and myc-tag. GST-SKP1 was a kind gift from Ashutosh Kumar (IIT Bombay, India). Ubc13 was from kind gift from Dr Ranabir Das (NCBS, India). His-ubiquitin and K63 (ubiquitin mutant has only K63 lysine, rest lysine residues are mutated to alanine)-only ubiquitin mutants were the kind gift from Dr Wuhan Xiao (Chinese Academy of Science, China). cDNA of β-TrCP1 was a kind gift from Jae U. Jung (University of Southern California, USA). β-TrCP1 was subcloned in p3XFLAG-CMV-14 and pCMV-myc-C vectors. FLAG-F-box deleted β-TrCP1 (ΔF-β-TRCP1), S158A-β-TrCP1 and S158D-β-TrCP1 were cloned in p3XFLAG-CMV-14 vector (Sigma). FLAG-β-TrCP2 and FLAG-F-box deleted β-TrCP2 (FLAG-ΔF-β-TrCP2) were also cloned in p3XFLAG-CMV-14 vector (Sigma). β-TrCP2 and degron mutant-β-TrCP2 (DM) were also cloned in pCMV-myc-C vector. β-TrCP1 and β-TrCP2 were also cloned in PET-28a vector. Primers used in this study are listed in Table S1 . Short hairpin RNAs (shRNAs) and packaging plasmids (pPAX2 and pMD2.G), GST-MDM2 and GST-MDM2 (C464A) were kind gifts from Prof. Michael R. Green (University of Massachusetts Medical School, USA). shRNAs sequences are mentioned in Table S2 .
3
Metabolic Reprogramming of SHSY5Y and 293LTV Cells
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SHSY5Y cells were purchased from ECACC (94030304) and cultured in DMEM containing 4 mM glutamine and 5.5 mM glucose, or 10 mM galactose, respectively. 293LTV cell line was purchased from Cell Biolabs, Inc. (LTV-100) and cultured in DMEM containing 4 mM glutamine and 5 mM glucose. The GCN5L plasmid was a kind gift from Dr. Sack’s laboratory19 (link). The IDH2 WT and IDH2 K413Q plasmids were a kind gift from the laboratory of Dr. Denu13 (link). Stable cell lines were generated by treatment with G418 (200 µg/ml) after transfection. SIRT3 and SIRT3 H248Y plasmids were purchased from Addgene (#24924, #24917 respectively) and respective ORFs subcloned into the p3XFLAG-CMV14 vector (Sigma-Aldrich). Vector DNA was transfected using Lipofectamine 2000 (ThermoFisher Scientific). For RNA silencing, the predesigned Mission siRNAs (Sigma-Aldrich) were transfected using Lipofectamine RNAiMAX (ThermoFisher Scientific). The decline of RNA was verified using real-time PCR and data calculated using Double Delta Ct analysis.
4
Generating Doxycycline-Inducible Mutant IDH Cell Lines
5
Construction of miR-92a Expression Vector
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To construct the mouse miR-92a-expressing vector, the primary sequence was the polymerase chain reaction (PCR), amplified and cloned into HindIII/EcoRV sites of the p3XFLAG-CMV14 vector (Sigma). The genomic element containing the c13orf25 promoter was PCR amplified and cloned into HindIII/EcoRV sites of the pGL3-Basic vector (Sigma). To construct the pRL-mRBM4a reporter, the coding element of mouse RBM4a, containing the putative targeting sites of miR-92a, was PCR-amplified and then inserted into Xba I/Not I sites of the pRL-TK vector (Promega). The derived mutant plasmids harboring substituted nucleotides were all constructed using the QuikChange site-directed mutagenesis system (Stratagene, La Jolla, CA, USA).
6
MTHFD2 Mutations for siRNA Resistance
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Human MTHFD2 complementary (c)DNA with point mutations to confer resistance against three MTHFD2 siRNA oligonucleotides (J-009349-10, J-009349-11 and J-009349-12) was obtained from DNA technology and ligated into a p3xFLAG-CMV14 vector (Sigma-Aldrich). To generate MTHFD2 Q132K:D155A, GeneArt Site-Directed Mutagenesis PLUS kit was used on the siRNA-resistant MTHFD2 cDNA to introduce point mutations. All sequences were verified by sequencing (KI gene).
7
EGF Overexpression in Mouse Ears
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The 3X FLAG human (h) full‐length EGF vector was constructed by inserting the cDNA to the p3xFLAG‐CMV14 vector (Sigma Chemical Co., Saint Louis, MO). Flag‐h EGF DNA plasmid driven by a CMV14 promoter (0.5 μg/ml) (EGF‐expression vector) was transfected once (n = 2, 4 ears) or five times every fourth day into the epithelial region of the mouse ear canals (n = 12, 12 ears) using a Nepa21 Electroporator (Nepa Gene Co., Chiba, Japan) under anesthesia using a combination anesthetic (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol), according to the protocol of a previous study.25 As a control, a null plasmid driven by a CMV14 promoter (0.5 μg/ml) (empty vector) was transfected into the other ears (n = 12, 12 ears) of the mice.25
8
Molecular Cloning of ASK1 and Associated Proteins
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Human MAP3Ks, ELOB/C, CUL5 and CBFβ genes were amplified from the Mammalian Gene Collection complementary DNA (cDNA) library and subcloned into pcDNA-based vectors (Life Technologies, Gaithersburg, MD). The accession codes for the genes used are listed in Supplementary Table 1 . Haemagglutinin (HA)-tagged human ASK1 expression vector and its deletion mutants (ΔN, ΔC, NT, KD, CT and K709M) have been described previously48 (link)49 (link)50 (link)51 (link). The ASK1 cDNAs were inserted into the pcDNA4/HisMax vector (Life Technologies) to obtain Xpress (XP)-tagged ASK1 or into the pRetroX-TRE3G vector (Clontech, Palo Alto, CA) to generate retroviral vector. Plasmids encoding HIV-1NL4-3 Vif52 (link)53 (link), human A3G54 (link) and ubiquitin55 (link) have been described. The Vif mutants were generated with PCR-based molecular cloning procedures. HIV-1NL4-3 Nef was amplified from a pNL4-3 molecular clone56 (link) and subcloned into p3xFLAG-CMV-14 vector (Sigma-Aldrich, St Louis, MO). For in vitro protein synthesis, each cDNA was subcloned into pEU vector (CellFree Sciences, Ehime, Japan).
9
Detailed Plasmid Construction and Cloning
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pCMV-myc-FBXO31 plasmid was kindly provided by Prof. David F. Callen (University of Adelaide, Australia). DDK-FBXL20 was purchased from Origene. His-Ub, HA-PUMA, HA-ΔBH3-PUMA, HA-BAX, EGFP-BAX, and EGFP-BAX (S184A) were purchased from Addgene. GST-PUMA plasmid was kindly provided by Dr Nam-Chul Ha (Seoul National University, Republic of Korea). PUMA cDNA was cloned in p3XFLAG-CMV-14 vector (Sigma) at BamHI site. FLAG-PUMA (S10A) mutant was generated by site-directed mutagenesis. F-box motif deleted FBXL20 mutant (DDK-ΔF-FBXL20) was generated by using FBXL20 as a template, and PCR product was inserted into SgfI and MluI site of the pCMV6-entry vector. FBXL20 truncations (1–138, 135–240, 241–350, and 350–436 aa) were PCR amplified and cloned into pET21a vector (EMD Biosciences). List of all primer sequences used in cloning is given in Table S1
10
Constructing Mouse Genomic Reporters
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Coding regions of human MAP4K4 and SRSF3 were amplified using a polymerase chain reaction (PCR) with a mice fetal complementary (c)DNA library as the template. The PCR product was digested with Hind III/EcoR I and EcoR I/EcoR V restriction enzymes and inserted into the p3XFLAG-CMV14 vector (Sigma, St. Louis, MO, USA). The mouse genomic element containing SRSF3 exon 3, intron 3, and exon 4 was PCR-amplified with genomic DNA prepared from C3H10T1/2 cells as the template. The PCR product was digested with EcoR V and Sac I restriction enzymes, and then the insert was placed into the pUC19 vector (NEB). The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB). PCR-amplified fragments were digested with EcoR I and Hind III restriction enzymes prior to DNA ligation. Derived mutants of the expressing vectors or minigene reporters harboring substituted nucleotides were constructed using the QuikChange site-directed mutagenesis system (Stratagene, Amsterdam, The Netherlands). The vector-based short hairpin RNA targeting mouse SRSF3 was purchased from the RNAi core facility at Academia Sinica (Taipei, Taiwan).
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