The BDNF gene from codons 155 to 273 representing its functional domain was cloned and sequenced. The BDNF gene was amplified by the polymerase chain reaction (PCR) using the 5′-end PCR primers: 5′-GGATCCCACTCCGACCCCGCCCG-3′ and 5′-AAGCTTTCTTCCCCTTTTTAA TGGTCAGT-3′, coupled with the restriction sites for BamHI and Hind III respectively. The pSV-β-galactosidase control vector (Promega, Madison, WI, USA) was cut using the Hind III-NcoI restriction enzyme pair, and the CMV promotor isolated by Hind III digestion of the pGEM base (Promega) was inserted into the pSV-β-galactosidase control vector. The product was then digested with the BsaA I (New England BioLabs, Ipswich, MA, USA) and Ava I (New England BioLabs) restriction enzymes, and the amplified BDNF gene was inserted. The hypoxia-responsive element (P18 × 3) with the P18 sequence TGTCACGTCCTGCACGAC was inserted into the Sal I site of the pSV-β-galactosidase control vector. The constructed BDNF pDNA was subsequently transformed to the Escherichia coli JM 109 cells (ECOSTM 9-5; Yeastern Biotech, Taipei, Taiwan). The positive clones were selected by quick plasmid preparation and enzyme digestion, which were then verified by DNA sequencing.
Psv β galactosidase control vector
Manufactured by Promega
Sourced in United States
The PSV-β-galactosidase control vector is a plasmid used as a control in various molecular biology applications. It contains the gene encoding the β-galactosidase enzyme, which is commonly used as a reporter or marker in assays. The vector can be used to monitor and validate experimental procedures involving gene expression and reporter systems.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Luciferase assay kit, by Promega (5 mentions)
Luciferase assay system, by Promega (4 mentions)
β galactosidase enzyme assay, by Promega (4 mentions)
Pgl3 basic vector, by Promega (3 mentions)
β galactosidase enzyme assay system, by Promega (3 mentions)
Luciferase activity assay, by Promega (3 mentions)
Bsaai, by New England Biolabs (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Bacteriological agar, by Merck Group (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (1 mentions)
Basal medium eagle, by Merck Group (1 mentions)
Sfrp1 vector, by OriGene (1 mentions)
Pcmv5 smad2 ha, by Addgene (1 mentions)
Pcmv5b flag smad3, by Addgene (1 mentions)
Lipofectamine, by Promega (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
26 protocols using psv β galactosidase control vector
1
Cloning and Sequencing of BDNF Functional Domain
2
Transcriptional Regulation by KLF4 Isoforms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Promoter fragments of the human FGF3 and TRH genes were cloned into pGL3-Basic vector (Promega) before the luciferase gene. Mini- and microsatellites DNA fragments were cloned immediately after the luciferase gene in construct containing minimal FGF3 promoter (−236 - +84 bp from TSS). All transfections into HEK293 cells were done using Invitrogen™ Lipofectamine™ LTX Reagent with PLUS™ Reagent (15-338-030, Fisher Scientific) according to the product’s instructions. Luciferase reporter constructs were co-transfected with p3XFLAG-CMV™-7.1 vector (E7533, Sigma-Ardrich) expressing human KLF4 or KLF4K409Q proteins. Luciferase activity was measured 48 h post-transfections using Thermo Scientific™ Pierce™ Firefly Luciferase Glow Assay Kit (PI16177, Fisher Scientific) or Luciferase Reporter Gene Assay (11814036001, Sigma-Aldrich). Transfection efficiencies were normalized on co-transfected pSV-β-Galactosidase Control Vector (E1081, Promega). β-Gal levels were measured using β-Gal Reporter Gene Assay (11758241001, Sigma-Aldrich). Both reporters were quantified on the Cytation5 plate reader (BioTek). pGL3-Basic plasmid without the promoter served as a negative (no promoter) control. Its activity was set as 1 and promoter activities in all samples were calculated as fold increase over the “no promoter” sample. Error bars represent StDev from at least four independent experiments.
3
Investigating Epigenetic Modulations
4
Regulation of Wnt Signaling Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
sFRP1 vector was purchased from OriGene Technologies, Inc. (Rockville, MD, USA). The green fluorescent protein-fused wild-type (WT) Rac1, constitutively active (CA) mutant Rac1 (Q61L), dominant-negative (DN) mutant Rac1 (T17N) (27 (link)), Tag5Amyc-GSK3β WT (28 (link)), pCS2 Flag Smad3 S204A (29 (link)), pCMV5B-Flag-Smad3 (30 (link)) and pCMV5 Smad2-HA (31 (link)) were purchased from Addgene, Inc. (Cambridge, MA, USA). Top-flash luciferase plasmid (BPS Bioscience, San Diego, CA, USA) is a luciferase reporter plasmid that contains two sets of 3 copies of the wild-type T-cell factor (TCF) binding regions. If the canonical Wnt signaling is activated, β-catenin will translocate to the nucleus to associate with TCF/lymphoid enhancer factor transcription factors to activate transcription of Wnt target genes. pSV-β-Galactosidase control vector was used as an internal control for transfection and was purchased from Promega Corporation (Madison, WI, USA). For plasmid transfection, SGC-7901/vector cells were seeded into 6-well plate and allowed to grow 24 h prior to transfection. Cells in each well were transfected with 4 µg plasmid using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h at 37°C and the medium was changed subsequent to transfection.
5
Transient Transfection Luciferase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transient transfection of luciferase reporter plasmids was performed using lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Cells were plated in 12-well tissue culture plates at 2 × 105 per well for overnight. Nucleic acid-lipofectamine 2000 mixtures containing 0.5 μg of luciferase reporter vectors, 0.5 μg of pSV-β-galactosidase control vector (Promega) and 0.5 μg of expressing vectors or 40 nM siRNA were exposed to cells. After 24 h, cell lysates were harvested and assayed for the Luciferase activity assay (Promega), performed according to the protocol recommended by the supplier. To normalize the transfection efficiency, the same cell lysates were also assayed for β-galactosidase activity using the β-Galactosidase enzyme assay (Promega).
6
Detecting β-Catenin Response Elements
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect β-catenin response elements (RE) activities in AP- or NP-treated cells (n = 3 in each condition), transfections of luciferase reporter plasmids, and pSV-β-galactosidase control vector (Promega Corp., Madison, WI, USA) were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After incubation for 6–12 h, the cells were rinsed with PBS and were lysed in 100 μl of reporter lysis buffer (Promega Corp., Madison, WI, USA). The mean normalized luciferase activity was taken [19] (link).
7
Investigating Signaling Pathways in Cellular Models
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit polyclonal antibodies specific for p-MEK and p-mTOR were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies specific for BDNF, MEK, p-ERK, ERK, mTOR, VEGF-C and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LYEV-1 antibody was purchased from Abcam (Cambridge, MA, USA). Recombinant human BDNF was purchased from R&D Systems (Minneapolis, MN, USA). ON-TARGETplus siRNAs were purchased from Dharmacon Research (Lafayette, CO, USA). The miR-624-3p mimic, miRNA control, Lipofectamine 2000, and Trizol were purchased from Life Technologies (Carlsbad, CA, USA). DMEM, α-MEM, fetal bovine serum and all other cell culture reagents were purchased from Gibco-BRL life technologies (Grand Island, NY, USA). The BDNF-shRNA plasmids were purchased from RNAiCore (Taipei, Taiwan); their sequences are provided in Supplementary Table S1 . The pSV-β-galactosidase control vector and luciferase assay kit were purchased from Promega (Madison, WI, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
8
NFAT Transcriptional Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transient transfection of NCTC cells was carried out using Lipofectamine 2000 Transfection Reagent (Life Technologies) according to the manufacturer's protocol. For siRNA transfection, cells were incubated with 60 nM of siRNA pool targeting either T-plastin or NFAT2 (Thermoscientific) for 24 hours before scratching assays or RNA and protein extraction. As control siRNA, a non-relevant siRNA sequence pool (Thermoscientific) was used.
In order to assess NFAT transcriptional activity, cells were transfected with PGL4 Luciferase Reporter Vector containing NFAT response element (NFAT-Luc vector, Promega) (2 µg) in association or not with siRNA and were co-transfection with β-galactosidase reporter vector (pSV-β-Galactosidase Control Vector, Promega) (0.2 µg) for normalisation of luciferase activity. In some cases, cells were pre-treated with FK506 for 30 minutes. Twenty four hours after transfection, cells (1.106) were harvested by trypsinisation, washed twice with PBS, and lysed with Reporter Lysis Buffer before luciferase assay detection (Luciferase Assay System, Promega) and assessment of β-galactosidase activity using the β-Galactosidase Enzyme Assay System (Promega). All the assays were performed in duplicate.
In order to assess NFAT transcriptional activity, cells were transfected with PGL4 Luciferase Reporter Vector containing NFAT response element (NFAT-Luc vector, Promega) (2 µg) in association or not with siRNA and were co-transfection with β-galactosidase reporter vector (pSV-β-Galactosidase Control Vector, Promega) (0.2 µg) for normalisation of luciferase activity. In some cases, cells were pre-treated with FK506 for 30 minutes. Twenty four hours after transfection, cells (1.106) were harvested by trypsinisation, washed twice with PBS, and lysed with Reporter Lysis Buffer before luciferase assay detection (Luciferase Assay System, Promega) and assessment of β-galactosidase activity using the β-Galactosidase Enzyme Assay System (Promega). All the assays were performed in duplicate.
9
Survivin Luciferase Assay in HCT116 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HCT116 cells (2 × 105 per well of a 6-well plate) were transiently transfected with 1 μg of the pGL3-survivin-luciferase construct and pSV-β-galactosidase control vector (Promega) using Lipofectamine. After transfection for 24 h, HCT116 cells were treated with 10 μM of IWR-1 for 24 h, and then harvested using the lysis buffer. Luciferase activity in cell extracts was measured using the Dual-luciferase assay kit (Promega) and a luminometer (Berthold technologies, Bad wildbad, Germany); it was normalized for transfection efficiency using the β-galactosidase assay (Promega).
10
Wnt Signaling Pathway Assay in HEK293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SuperTopFlash (STF) construct (generously provided by Dr. Amir Rattner and Dr. Jeremy Nathans of Johns Hopkins University) contains a firefly luciferase reporter driven by seven lymphoid enhancer factor/T cell factor proteins (LEF/TCF) consensus binding sites. LEF/TCFs mediate Wnt signals in the nucleus by recruiting β-catenin to Wnt target genes. This reporter plasmid was stably transfected into HEK293 cells as reported previously to generate the STF cell line [19 (link)]. The STF cells were cotransfected with 200 ng of Norrin, 200 ng of FZD4, 240 ng of LRP5 (wild type or mutant), and 100 ng of pSV-β-Galactosidase Control Vector (Promega, Madison, WI) in a 24-well plate using Lipofectamine™ 2000 Transfection Reagent (Invitrogen, Carlsbad, CA). The transfected cells were washed with PBS twice after 48 h of transfection and assayed using the Promega Luciferase reporter assay system. The firefly luciferase activity was normalized to the coexpressed β-galactosidase activity. Each assay was performed in triplicate at the same time and repeated three times.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!