Retroviral particles encoding a self-excising Cre recombinase33 (link), full-length E4F1WT, or its E3 ligase (Δ41–84, referred to as E4F1ΔE3) or p53 binding (Δ520–569, referred to as E4F1Δp53) mutants were produced in 293 T packaging cells by transient transfection using Jet-PEI reagent (Ozyme). In all, 72 hrs after transfection, viral supernatants were harvested and added on Mefs overnight in presence of polybrene (5 μg/ml, Sigma). Antibiotic selection was performed 48 h later after transduction with hygromycin (50 μg/mL, Invitrogen). SCD1 and GFP adenoviruses were purchased from Vector Biolabs. Adeno-Cre was purchased from the University of Iowa’s viral core facility.
Jet pei reagent
Manufactured by Ozyme
Sourced in France
Jet-PEI is a transfection reagent used for the delivery of nucleic acids, such as plasmid DNA or siRNA, into mammalian cells. It is a cationic polymer that forms compact complexes with nucleic acids, facilitating their uptake by cells.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Turbofect, by Thermo Fisher Scientific (1 mentions)
Lipofectamin 2000, by Thermo Fisher Scientific (1 mentions)
P53 antibody do 1, by Santa Cruz Biotechnology (1 mentions)
Six well plate, by BD (1 mentions)
5 protocols using jet pei reagent
1
Retroviral Transduction of Mouse Embryonic Fibroblasts
2
Cell Culture and Transfection Protocol
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HEK293 and NIH3T3 were cultured in DMEM medium while BEAS-2B cells were cultured in LHC-8 medium, all supplemented with 10% fetal calf serum (Life Technologies, Carlsbad, CA, USA). BEAS-2B cells were transfected with TurboFect (Thermo Fisher Scientific, Waltham, MA, USA), HEK293 cells with Lipofectamin 2000 (Life Technologies) and NIH3T3 with JetPei reagent (Ozyme, St Quentin en Yvelines, France).
3
Analyzing p53 Transcriptional Activity
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H1299 cells were seeded (200 000 cells per well) into a six-well plate (Falcon) and 24 h later co-transfected—in the presence of the Jet Pei reagent (Ozyme), as recommended by the manufacturer—with pCMVp53WT or its mutant derivatives (3 μg), variants of p53BS-luc (2 μg) and pCMLacZ (0.6 μg). Media was exchanged six hours after transfection. The antibiotic G418 (200 μg/ml) was added with the fresh media 6 h post-transfection. Protein extracts were prepared 24 h after transfection and enzymatic activities were measured as previously described (46 (link)). For each experimental set-up, at least five independent transfection experiments were performed. For visualization of short and long forms of the p53 protein, protein extracts were prepared using 200 μl of a room tempered 1× Glo buffer; incubation for 5 min at 25°C while shaking at 550 rpm. Approximately 0.5 μg of protein extracts were subjected to the ‘Western-like’ analyses using the Jess Protein Simple device; the total protein was measured by Bradford. The p53 antibody (DO-1; the N-terminal epitope mapping between amino acid residues 11–25 of p53; Santa Cruz Biotechnology) was used at its saturating conditions 1:100. The equal loading was monitored by the total protein staining (Protein Simple).
4
BRCA1-Deficient Cell Line for HR Assay
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Experiments were carried out in RG37-shBRCA1 cell lines. RG37 cells are SV40-transformed human fibroblasts containing a chromosomally integrated DR-GFP substrate that specifically monitors gene conversion. RG37 cells were infected with lentiviral particles containing an shRNA directed against the 3′ UTR part of the BRCA1 messenger and the puromycin resistance gene. Puromycin was added 3 days after infection and cells were reseeded at low density to isolate individual clones. Clones were then screened for deficiency in the formation of BRCA1 foci after ionizing radiation, deficiency in HR (using the DR-GFP substrate), and for the extinction of BRCA1 expression monitored by Western blot analysis. The new cell line was called RG37-shBRCA1.
Cells were pretreated (or not) with 10 mg/mL of doxycycline 3 days before plating. They were then seeded at 2 × 105 per well in six-well plates. Twenty-four hours after plating, expression vectors encoding for BRCA1 (or its variant forms) and the HA-tagged meganuclease I-SceI were cotransfected using JetPEI reagent (PolyPlus, Ozyme). Forty-eight hours after transfection, cells were trypsinized and GFP + cells were directly measured by flow cytometry.
Cells were pretreated (or not) with 10 mg/mL of doxycycline 3 days before plating. They were then seeded at 2 × 105 per well in six-well plates. Twenty-four hours after plating, expression vectors encoding for BRCA1 (or its variant forms) and the HA-tagged meganuclease I-SceI were cotransfected using JetPEI reagent (PolyPlus, Ozyme). Forty-eight hours after transfection, cells were trypsinized and GFP + cells were directly measured by flow cytometry.
5
Knockdown and Overexpression of αII-Spectrin
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Cells were transfected with short hairpin RNA plasmids (shRNA, SA Biosciences) expressing GFP and a non-targeting sequence used as control (non-relevant shRNA, named Nr-shRNA) or a sequence targeting the αII-spectrin gene, (αII-spectrin shRNA named Sp-shRNA) using JET PEI reagent (Ozyme-Polyplus), according to the manufacturer’s instructions. Transfection efficiency was determined by flow cytometry (GFP expression) 24 hr after transfection. Four Sp-shRNA plasmids were tested: for mice cells referred as 1m, 2m, 3m and 4m and for human cells referred as 1 h, 2 h, 3 h and 4 h).Depletion efficiency of αII-spectrin expression was estimated by western blot at 24, 48, 72 and 96 hr after transfection. Other plasmids were also used to transfect MEF v-Src Y527F cells: recombinant full-length of αII-spectrin fused to GFP from pCep4 plasmid (pCep4 GFP αII-spectrin, generous gift from Dr Gaël NICOLAS), and plasmids expressing the LifeAct peptide fused with fluoro-Ruby (Ruby-LifeAct, a red marker visualising F-actin in living cells), β3-integrin tagged with RFP (clone 285, β3-Integrin RFP), actin tagged with RFP(RFP-actin), cortactin tagged with RFP (clone 145, RFP-Cortactin), or paxillin tagged with RFP (called RFP-paxillin in experiments), these sixth last allowing detecting invadosome structures.
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