The human OTUD7B cDNA (accession NM_020205.4 ) was cloned into the lentivirus vector pLenti-CMV-EGFP-3Flag to create the GFP or GFP-OTUD7B expression vector, and the OTUD7B cDNA sequence was confirmed by sequencing. LV3 lentiviral vectors encoding shRNAs silencing OTUD7B or a non-silencing control shRNA were purchased from GenePharma (Suzhou, China). The sequences of OTUD7B shRNAs: shOTUD7B#1: TTCTCCGAACGTGTCACGT; shOTUD7B#2: GCTGCGGAAAGCTTTGTATGC.
Lv3 lentiviral vector
Manufactured by GenePharma
Sourced in China
The LV3 lentiviral vector is a laboratory tool used for gene delivery and expression studies. It functions as a viral vector, providing a vehicle to efficiently introduce genetic material into target cells. The LV3 vector is derived from the human immunodeficiency virus (HIV) and is designed to safely and effectively transduce a variety of cell types. However, a detailed description of its intended use or performance characteristics is not available.
Automatically generated - may contain errors
Lab products found in correlation
Lv5 lentiviral vector, by GenePharma (3 mentions)
Polybrene, by Merck Group (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Puromycin, by MP Biomedicals (1 mentions)
Effective sirna oligonucleotides, by GenePharma (1 mentions)
Penter vector, by Charles River Laboratories (1 mentions)
Effective sirna oligonucleotides, by RiboBio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Ha ub, by Addgene (1 mentions)
Myc ulk1, by Addgene (1 mentions)
8 protocols using lv3 lentiviral vector
1
Generating OTUD7B Expression Vectors
2
Bclaf1 Knockdown and Overexpression in SW-13 Cells
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Effective siRNA oligonucleotides that target Bclaf1 were purchased from GenePharma (Suzhou, China). Full-length Bclaf1-overexpressing plasmids were kindly provided by Dr. Tang (College of Veterinary Medicine, China Agricultural University). SW-13 cells were transfected with oligonucleotides or plasmids using Lipofectamine 3000 (Invitrogen, CA, USA) and were harvested for assays 48 h after transfection. The synthesized short hairpin RNAs targeting Bclaf1 (shBclaf1) or nonspecific control RNAs (shNC) were designed and cloned into LV3 lentiviral vectors, which were constructed by GenePharma (Suzhou, China). The siRNA and shRNA sequences are listed in Table S1
3
Knockdown of DANCR in NPC Cells
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Effective siRNA oligonucleotides that targeted DANCR, NF45 or NF90 were purchased from RiboBio (Guangzhou, China). LV3 lentiviral vectors that encode short hairpin RNA (shRNA) targeting DANCR or non-specific control were constructed by GenePharma (Suzhou, China). The siRNA and shRNA sequences are shown in Table S1 . The pENTER-vector, pENTER-DANCR and pENTER-HIF-1α plasmids were purchased from Vigene Biosciences (Jinan, China). All of the plasmids were confirmed through DNA sequencing.
The synthesized short hairpin RNA targeting DANCR (shDANCR) or control shRNA were cloned into pSuper-retro-puromycin vectors (Addgene, Cambridge, MA, USA). NPC cells were transfected with oligonucleotides (100 nM) or plasmids (2 μg) using Lipofectamine 3000 or Lipofectamine 2000 reagents (Invitrogen). The cells were harvested for assays 48 h after transfection. SUNE-1 cell lines stably expressing shDANCR or vector were generated after lentiviral infection by 293FT cells and selected by 0.5 μg/mL puromycin.
The synthesized short hairpin RNA targeting DANCR (shDANCR) or control shRNA were cloned into pSuper-retro-puromycin vectors (Addgene, Cambridge, MA, USA). NPC cells were transfected with oligonucleotides (100 nM) or plasmids (2 μg) using Lipofectamine 3000 or Lipofectamine 2000 reagents (Invitrogen). The cells were harvested for assays 48 h after transfection. SUNE-1 cell lines stably expressing shDANCR or vector were generated after lentiviral infection by 293FT cells and selected by 0.5 μg/mL puromycin.
4
Lentiviral Overexpression and Knockdown of HHIP-AS1
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The full-length human HHIP-AS1 constructs were created following standard protocols and verified by gene sequencing. The cDNA of HHIP-AS1 was subcloned into the LV5 lentiviral vector (GenePharma Company, Suzhou, China) for overexpression. Subsequently, the LV3 lentiviral vector (GenePharma) was used to construct a short hairpin RNA (shRNA) of HHIP-AS1. The PDLSCs were seeded and cultured in 100 mm dishes overnight and infected with lentiviruses and 6 μg/mL polybrene (Sigma-Aldrich) for 12 h. Then, the transfected PDLSCs were selected by proper antibiotics after 72 h of infection. The target sequences of the shRNAs were as follows: control shRNA (Consh), 5′-TTCTCCGAACGTGTCACGTTTC-3′, and HHIP-AS1 shRNA (HHIP-AS1sh), 5′-GCACCAATGCATCTTGTATGA-3′.
5
Lentiviral Expression of Human XR-111050 in MSCs
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The plasmids were constructed with standard methods; all structures were verified by appropriate restriction digestion and/or sequencing. Human full-length XR-111050 cDNA from BMSCs was produced with a standard PCR protocol. This sequence was subcloned into the LV5 lentiviral vector (Genepharma Company, Suzhou, China). Short-hairpin RNAs (shRNA) with the complementary sequences of the target genes were subcloned into the LV3 lentiviral vector (Genepharma Company, Suzhou, China). For viral infections, MSCs were plated overnight and then infected with lentiviruses in the presence of polybrene (6 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) for 6 h. After 48 h, infected cells were selected with 1 μg/mL puromycin for 7 days. The target sequences for the shRNA were: LV3 shRNA (Consh), 5’-TTCTCCGAACGTGTCACGTTTC-3’; XR-111050 shRNA (XR-111050sh), 5’-GGACGTGTCTTTCAGGGAAAG-3’.
6
Modulation of TRAF and ULK1 Signaling
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Expression vectors encoding human TRAF2 and TRAF3 and truncation mutants of TRAF3 (88-C, 333-C, and 1-448), myc-ULK1, Flag-cIAP1, Myc-cIAP1 H588A, and HA-Ub were purchased from Addgene. pGIPZ Lentiviral vectors encoding shRNAs specific targeting human ULK1 or a nontargeting control shRNA (shNT) were purchased from GE healthcare (CO, USA). Lentiviral vectors silencing human TRAF3 were purchased from Sigma-Aldrich. LV3 lentiviral vector silencing human TRAF2 and LV10 lentiviral vector silencing human AIF were purchased from GenePharma (Suzhou, China). The sequences of shRNAs listed in Table 1. All DNA constructs used were verified by DNA sequencing.
7
HOTTIP and miR-574-5p Modulation in SCLC
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HOTTIP small interfering (si)RNA and miR-574-5p inhibitors or mimics (Tables I and II ) were obtained from Suzhou GenePharma Co., Ltd. The synthesized HOTTIP and miR-574-5p interference sequences or plasmids as well as their corresponding NC sequences were transfected into SCLC cells, and the cells with transient down-regulation of HOTTIP and miRNA were obtained. Briefly, 8–10×105 cells were inoculated and cultured in a 6-well plate at 37°C in a 5% CO2 incubator. The next day, whe the confluence 70–80%, Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific, Inc.) was used to transfect 10 nM plasmid or 40 nM siRNA into 1×106 cells at 37°C for 4–6 h. Subsequently, the medium containing the transfection reagent was discarded and the complete medium was replaced. After 24–48 h, RNA or protein was extracted for subsequent experiments. The lentiviral method was used for stable transfection; the most effective interference sequence of HOTTIP (si-h-HOTTIP-1, confirmed by RT-qPCR) was packaged in an LV3 lentiviral vector (Shanghai GenePharma Co.) for target cell infection experiment, LV3-NC was used as a control, and SCLC cell lines stably silencing HOTTIP were used for further experiments.
8
Lentiviral-Mediated Overexpression and Knockdown
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The plasmids used in this study were constructed with standard protocols; all constructs were verified by gene sequencing. The cDNA of human DLX5 or HOXC8 were subcloned into the LV5 lentiviral vector (Genepharma Company, Suzhou, China) separately for over-expression in SCAPs. Short hairpin RNAs (shRNAs) of DLX5, LINC01013, or HOXC8 was subcloned into the LV3 lentiviral vector (Genepharma). Then, SCAPs were plated and cultured for overnight in 100-mm dish and infected with lentiviruses for 12 h, containing polybrene (6 μg/ml, Sigma-Aldrich, St. Louis, MO, USA). The fresh medium was replaced after 48 h, and the transfected SCAPs were selected with the appropriate antibiotics. The target sequences for the shRNAs were as follows: HOXC8 shRNA (HOXC8sh), 5′-GGAGACGCCTCCAAATTCTAT-3′; DLX5 shRNA (DLX5sh), 5′-GTGCAGCCAGCTCAATCAA-3′; LINC01013 shRNA (LINC01013sh), 5′-GGTAATGACTGAGGTTATTCC-3′; and control shRNA (Consh), 5′-TTCTCCGAACGTGTCACGTTTC-3′.
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