The following plasmids were constructed and purchased from GeneChem (Shanghai, China): plasmids (CMV-MCS-3FLAG-SV40-neomycin) containing the full length of human MLKL coding sequence (gene ID: 197,259) and its mutant form (gene ID: 197,259 (S454A)) or control plasmids; the plasmids (CMV-MCS-Myc-SV40-neomycin) containing the full length of human GJA1 coding sequence (gene ID: 2697) and its mutant form (gene ID: 2697 (K303G)) or control plasmids; the plasmids (CMV-MCS-HA-SV40-neomycin) containing human Ub coding sequence (gene ID: 7316); and the plasmids (CMV-MCS-V5-SV40-neomycin) containing human VHL coding sequence (gene ID: 7428). TurboFect Transfection Reagent (Thermo, Cat# R0531) and plasmids were prepared in the following ratios: plasmid (1 μg): TurboFect Transfection Reagent (3 μL). After 20-minute incubation, the mixture was added to the cells seeded in the dish and left for 48 h for further analysis, with appropriate replacement of fresh medium. The final concentration of plasmid was 0.8 μg/mL.
Cmv mcs 3flag sv40 neomycin
Manufactured by Genechem
Sourced in China
The CMV-MCS-3FLAG-SV40-neomycin is a plasmid vector used for gene expression and selection in mammalian cell lines. It contains a cytomegalovirus (CMV) promoter, a multiple cloning site (MCS), three FLAG epitope tags, the SV40 origin of replication, and a neomycin resistance gene for selection.
Automatically generated - may contain errors
Lab products found in correlation
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Rpmi opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
B27 and glutamax, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Lead acetate, by Merck Group (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Opti mem culture medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using cmv mcs 3flag sv40 neomycin
1
Plasmid Transfection in Cell Lines
2
Overexpression of GRP78, AR-V7, SIAH2 in cells
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The following plasmids were constructed and purchased from GeneChem: plasmids (CMV-MCS-3FLAG-SV40-neomycin) containing the full length of human GRP78 CDS (gene ID: 3309) and its truncated mutants or control plasmids; the plasmids (CMV-MCS-HA-SV40-neomycin) containing the full length of human AR-V7 CDS (gene ID: 367) and its mutant form that lacks ΔCE3 on its C-terminal (AR-V7ΔCE3) or control plasmids; and the plasmids (CMV-myc-MCS) containing human SIAH2 CDS (gene ID: 6478). Lipofectamine 3000 (Invitrogen) reagent, RPMI opti-MEM (Gibco), and the plasmids were prepared in the following ratio: P3000 (2 μl): plasmids (1 μg): Lipofectamine 3000 (2 μl). After incubation for 15 min, the mixtures were added to the cells seeded on plates or dishes and remained there for 48 h for further analysis, and fresh medium was replaced appropriately. The final concentration of the plasmids was 0.75 μg/ml.
3
Primary Neuron Culture and Lead Exposure
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Primary neurons were prepared from embryonic day18~19(E18~19) rat hippocampus as previously described by Craven et al.54 (link). Hippocampi were collected by dissection on ice and dissociated with scissors. 0.5 ml of 2.5% trypsin was added and incubate for 20 min in a water bath at 37 °C, Gently remove trypsin solution and triturated the hippocampi. Plate the desired number of cells (1.5 * 106 cells/ml) to dishes precoated with poly-L-lysine (0.5 mg/ml) (Sigma-Aldrich, USA) in serum-free neurobasal media supplemented with B27 and glutamax (GIBCOBRL, USA) then put it in incubator. At day in vitro (DIV) 3, add cytosine arabinoside (1-b-D-arabinofuranosylcytosine) to inhibit glial proliferation in a final concentration of 5 mM. Neurons were transfected with CMV-MCS-3FLAG-SV40-Neomycin (Genechem, Shanghai, China) at 4 mg per well for 6-well plates by Lipofectamine 2000 (Invitrogen) at DIV 6. Since during DIV 7–12 is the primary time period of dendritic spine growth55 (link), for lead exposure, cultures were treated with lead acetate (1 μM, Sigma-Aldrich, USA) for 5 days from DIV 7 to DIV 12.
4
Knockdown and Rescue of Orai1 in Cells
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Small hairpin RNA (shRNA) directed against Orai1 was generated using the GV112 vector (U6-MCS-CMV-puromycin) (GeneChem, Shanghai, China). The sequence used was 5′-CGTGCACAATCTCAACTCG-3′ [7 (link)]. Cells transfected with shOrai1 were selected using puromycin (5 μg/ml). The cDNA construct for re-expression of Orai1 was obtained by site-directed mutation of the targeting sequences without changing the amino acid sequence. The mutant was subcloned into a GV141 vector (CMV-MCS-3FLAG-SV40-Neomycin) (GeneChem, Shanghai, China). Cell stably transfected with shOrai1 were transfected with the Orai1 rescue construct and selected again using G418 (1 μg/ml). The shRNA plasmid for Pyk2 knockdown was purchased from Santa Cruz Biotechnology and was a pool of three target-specific 19–25-nt small interfering RNAs (siRNAs) designed to knock down gene expression. The control shRNA plasmid was also provided. Lipofectamine 2000 reagent (Invitrogen) was used for transfection of the Orai1 construct and shPyk2. The whole process was performed according to the manufacturer’s instructions.
5
CRTC2 Overexpression in Hepatic Cells
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The human hepatic cell lines HepG2, SK-Hep-1, LM3, and Huh7, obtained from the Chinese Center for Type Culture Collection, were cultured in DMEM (Life Technologies) containing 1.0 g/L glucose and 10% FBS at 37°C in a humidified atmosphere of 5% CO 2 . For CREB-regulated transcription coactivator 2 (CRTC2) overexpression in vitro, HepG2 and Huh7 cells were transfected with GV141 ligated to CMV-MCS-3FLAG-SV40-neomycin (Shanghai Genechem) in Opti-MEM culture medium (Gibco) using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's instructions, with GV141-empty used as control. The Opti-MEM was replaced by normal culture medium at 5 hours after transfection, and cells were harvested at 48 hours after transfection for the associated experiments.
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