The human ESCC cell line ECA109 (purchased from ATCC, Manassas, VA, USA) was cultured in RPMI-1640 (GIBCO, Waltham, MA, USA; 1.5 g/L NaHCO3, 2.5 g/L glucose, 0.11 g/L sodium pyruvate), with 10% fetal calf serum, at 37°C and 5% CO2. PCMV-ARHI-AC-GFP and PCMV-AC-GFP were purchased from OriGene (Rockville, MD, USA). The ARHI plasmid PCMV-ARHI-AC-GFP was transfected into ECA109 cells; ARHI was overexpressed in vitro and verified by western blotting. The empty plasmid PCMV-AC-GFP (OriGene) was used as a negative control. ARHI-targeted small interfering RNA (siRNA) was purchased from GenePharma (Shanghai, China), the sequence of target was: CUGCUUGACAAGUGCAUAATT. Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) was used for transfection according to the manufacturer’s instructions. After 48 h, the transfected cells were harvested and used for further experiments.
Pcmv ac gfp
Manufactured by OriGene
Sourced in United States
PCMV-AC-GFP is a plasmid vector designed for the expression of proteins tagged with green fluorescent protein (GFP) in mammalian cells. The plasmid contains the cytomegalovirus (CMV) promoter, which drives the expression of the GFP-tagged protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Eca109, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Pcdna3, by Addgene (1 mentions)
Pcmv mir, by OriGene (1 mentions)
Pgfp 5 rs, by OriGene (1 mentions)
T4 ligase, by Promega (1 mentions)
Anti turbogfp antibody, by Evrogen (1 mentions)
Mlu 1 enzyme, by New England Biolabs (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
4 protocols using pcmv ac gfp
1
Overexpression and Silencing of ARHI in ESCC
2
Hepatoma Cell Lines Hep3B and HepG2 Culture
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Human hepatoma cell lines Hep3B and HepG2 were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). These cell lines were maintained in Dulbecco's modified Eagle medium (Gibco BRL Life Technologies) supplemented with 10% fetal bovine serum (sigma) in a humidified atmosphere of 5% CO2 incubator at 37°C. pCMV-miR, pCMV-miR675 (MI0005416), pGFP-V-RS, pCMV-AC-GFP, pCMV6-entry-EGR1, pGFP-V-RS-EGR1, pMitTarget were purchased from Origene (Rockville, MD, USA) and pcDNA3.1, pcDNA3.1-HA--HP1α, pcDNA3.1-HA--HP1β, pcDNA3.1-HA--HP1γ, pBS-H19, pGL3-C-myc, were purchased from Addgene (Cambridge MA, USA). pCI-H19, pMirTarget-HP1α, β, γ3′UTR, pMirTarget-mutant HP1α, β, γ3′UTR, pCMV-mutant miR675, pGFP-V-RS-mir675, pGFP-V-RS-H19, pGFP-V-RS-PKM2, pGL3-EGR1, pGL3-H-Ras, pGL3-CyclinD1, pGL3-Pim1, pGL3-RB was constructed by ourselves.
3
Plasmid Construction and Western Blot
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PCR product was ligated into multiple cloning sites of pCMV-AC-GFP after digestion of restriction enzymes, Sgf I and Mlu I. pCMV-AC-GFP was purchased from ORIGENE (Catalog #PS100010), Sgf I enzyme from NEB (Catalog #R0630S), Mlu I enzyme from NEB (Catalog #R0198S), and T4 ligase from Promega (Catalog #M180A). We transfected HEK293 cells with constructed plasmid 48 h before collecting cells in lysis buffer. Western blot experiments were conducted using these cell lysates. Anti-TurboGFP antibody was purchased from Evrogen (Catalog #AB513).
4
Engineered DNM1 Variants for Cell Study
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Wild-type (WT) DNM1 cDNA was cloned into the pCMV-AC-GFP (OriGene, Rockville, MD) and pCMV-AC-RFP (OriGene) vectors in frame. Mutagenesis was performed on green fluorescent protein (GFP)-tagged DNM1 by site-directed mutagenesis using the NEB Q5 site-directed mutagenesis kit to create the c.529G>C (A177P), c.618G>C (K206N), and c.1076G>C (G359A) mutations according to the manufacturer's protocol. Plasmid sequences were verified by Sanger sequencing.
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