pGL3-HK2, pGL3-Bcl-2, pRL-TK-Renilla plasmid and pCF CREB, pCMV4 and pSV-SPORT expression vectors were gifts provided by Professor Dongping Wei (The First Affiliated Hospital of Nanjing, Nanjing, China). The mammalian expression vector pcDNA3.1 was purchased from Thermo Fisher Scientific (San Jose, CA, USA). The HK2 adenovirus and the control virus were purchased from Obio Technology Co., Ltd. (Shanghai, China). The pSV Sport PPARγ (Addgene plasmid no. 8886) and pCMV4 p65 (Addgene plasmid no. 21966) plasmids were gifted by Dr Bruce Spiegelman (Dana-Farber Cancer Institute, Harvard Medical School) and Dr Warner Greene (Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center), respectively.
Pcmv4 p65
Manufactured by Addgene
Sourced in United States
PCMV4-p65 is a plasmid vector that contains the CMV promoter and the p65 subunit of NF-κB. It is commonly used for the expression of the p65 protein in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Secrete pair dual luminescence assay kit, by GeneCopoeia (2 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Hk2 adenovirus, by Obio Technology (1 mentions)
Berberine, by Merck Group (1 mentions)
Alexa fluor 488 labeled goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Rabbit anti nf κb p65, by Abcam (1 mentions)
Rabbit anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Lipofectamine 3000 reagent, by Addgene (1 mentions)
Endofectin max transfection reagent, by GeneCopoeia (1 mentions)
V3lhs 364015, by Thermo Fisher Scientific (1 mentions)
V3lhs 409093, by Thermo Fisher Scientific (1 mentions)
Sirna oligos, by Integrated DNA Technologies (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 transfection reagent, by GeneCopoeia (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Egfpc1 hunfatc1ee wt, by Addgene (1 mentions)
6 protocols using pcmv4 p65
1
Plasmid Transfection and Adenovirus Infection
2
NF-κB p65 Activation and Apoptosis Regulation
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Berberine and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-NF-κB p65 and rabbit anti-caspase 8 antibodies were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-GAPDH antibody and Alexa Fluor® 488-labeled goat anti-rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were of analytical grade unless indicated otherwise. The NF-κB p65 expression vector pCMV4-p65 (plasmid #21966; Addgene) and empty vector (pCMV4-3HA; plasmid #24165; Addgene) were a gift from Dr. Warner Greene.
3
EZH2 Promoter Luciferase Assay
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The detailed procedure was reported previously [33 (link)]. Briefly, NSCLC cells were seeded at a density of 2.5 × 105 cells/well in 6-well dishes and grown to 60 % confluence. For each well, 2 μg of the control or wild type pEZX-PG04-EZH2 promoter constructs (purchased from GeneCopoeia, Inc., Rockville, MD, USA) with or without 0.2 μg of the internal control secreted alkaline phosphatase (SEAP) were co-transfected into the cells with the EndoFectin Max transfection reagent. The preparation of cell extracts and measurement of luciferase activities were determined using the Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia, Inc., Rockville, MD, USA), and was normalized with SEAP activity within each sample. In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34 (link)] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent. Cells were incubated for 24 h at 37 °C, then treated with PPI for the indicated time for all other experiments.
4
RRAD Overexpression and Knockdown in Lung Cancer
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Human lung epithelial cancer H1299 and H460 cells were purchased from ATCC (Manassas, VA). For cells with stable ectopic RRAD overexpression, cells were transduced with a pLPCX-RRAD-Flag retroviral vector and selected by puromycin [16 (link)]. The pCMV-RRAD-Flag WT and deletion mutations were constructed by PCR amplification. pCMV-p65-HA vector was constructed by using DNA fragment from pCMV4-p65 (Addgene). The lentiviral shRNA vectors against human RRAD (V3LHS_364015 and V3LHS_409093) were obtained from Open Biosystems (Huntsville, AL). To avoid off-target effects, two different siRNA oligos against each gene were employed for all knockdown experiments. The siRNA oligos against human GLUT1 (5′- CGAACTATGAACTACAAAGCTTCTA-3′ and 5′-TCAAAGTTCCTGAGACTAAA GGCCG-3′) and human p65 (5′-GGAGTACCCTGAGGCTATAACTCGC-3′ and 5′- AGCACAGATACCACCAAGACCCACC-3′) were obtained from Integrated DNA Technologies. Vectors and siRNA oligos were transfected into cells using Lipofectamine 2000 (Invitrogen).
5
Transfection and Luciferase Assay Protocol
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This procedure was reported previously66 (link). The control, MUC1 overexpression vectors were obtained from OriGene Technologies, Inc. (Rockville, MD, USA). The control and AMPKα expression vector (M02-AMPKα) were obtained from the GeneCopoeia, Inc. (Rockville, MD, USA). The control and p65 overexpression vector (pCMV4-p65) was obtained from the Addgene (Plasmid #21966)67 (link). Briefly, cells were seeded in 6-well dishes and grown to 50–60% confluence. For each well, 2 μg of control, MUC1, AMPKα and p65 plasmid DNA constructs were transfected into the cells using Lipofectamine 3000 Transfection Reagent (Invitrogen, Shanghai, China) for up to 24 h, followed by treating with solamargine for an additional 24 or 48 h. In the separated experiments, control and wild type pEZX-PG04-MUC1 promoter constructs (purchased from GeneCopoeia, Inc., Rockville, MD, USA) with or without 0.2 μg of the internal control secreted alkaline phosphatase (SEAP) were co-transfected into the cells with the Lipofectamine 3000 Transfection Reagent. The preparation of cell extracts and measurement of luciferase activities were determined using the Secrete-Pair™ Dual Luminescence Assay Kit (GeneCopoeia, Inc).
6
Lentiviral Constructs for Cellular Signaling
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LCAG-HBG, LEG11-RelA, and LEG11-NFAT2 lentiviral expression plasmids were created by Gibson Assembly cloning based on a split-GFP system described previously (Cabantous et al., 2005 (link), 2013 (link)). RelA or NFAT2 was PCR amplified from the pCMV4-p65 (#21966; Addgene; Ballard et al., 1992 (link)) or the EGFPC1-huNFATc1EE-WT (#24219; Addgene; Beals et al., 1997 (link)) plasmid. EF1α-ERK-KTR-mScarlet (LE-EKS), EF1α-JNK-KTR-mScarlet (LE-JKS), and EF1α-p38-KTR-mScarlet (LE-38KS) lentiviral expression vectors were generated by Gibson Assembly cloning based on the ERK-KTR-Clover, a JNK-KTR-mRuby2 and p38-KTR-mCerulean3 plasmids from the Markus Covert lab (#59150, #59154 or #59155; Addgene; Regot et al., 2014 (link)). TCR55α, TCR55β, TCR6α, and TCR6β chains were cloned individually into lentiviral pHR vectors based on previously reported constructs (Sibener et al., 2018 (link)). EF1α-human-PDCD1 (LE-hPDCD1) and EF1α-human-PD-L1 (LE-hPDL1) lentiviral expression vectors were constructed by Gibson Assembly cloning based on a human PD-1 cDNA template (HG10377-M; SinoBiological) or a human PD-L1 cDNA template (HG10084-M; SinoBiological). EF1α-human-CD80 (LE-hCD80) and UbC-human-CD86 (LU-hCD86) lentiviral expression vectors were obtained from Applied Biological Materials (#155690610695 and #155750610895 respectively).
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