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Pcdna5 backbone

Manufactured by Thermo Fisher Scientific
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The PcDNA5 backbone is a plasmid vector designed for high-level, tetracycline-inducible expression of recombinant proteins in mammalian cell lines. It contains the necessary elements for efficient selection, propagation, and expression of target genes in eukaryotic cells.

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Lab products found in correlation

Pmx backbone, by Cosmo Bio (2 mentions) Dynabeads protein g immunoprecipitation system, by Thermo Fisher Scientific (2 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions)

Close spelling variants of this product

PCDNA5 FRT/TO plasmid backbone PcDNA5/FRT/TO vector backbone

2 protocols using pcdna5 backbone

1

Immunoprecipitation of A20 and p62 Complexes

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Cells were stimulated with Pam3CSK4 (100 ng/ml) or zymosan (100 μg/ml) and lysed with RIPA buffer and NP-40 (for immunoprecipitation) containing protease inhibitor cocktail (P8340, Sigma). Phosphatase inhibitor cocktail II was added for p-IκB detection. To examine the association between A20 and p62, NIH-3T3 cells were co-transfected with expression vectors for A20 and p62 (pcDNA5-A20-V5/His and pMI-p62-HA, respectively) by using Lipofectamine LTX (Invitrogen), as recommended by the supplier. The expression vectors for A20 and p62 were made in our laboratory, as fusions of the V5/His tag in the pcDNA5 backbone (Invitrogen) and the HA tag in the pMX backbone (Cosmobio), respectively. Immunoprecipitation was carried out by using Dynabeads Protein G Immunoprecipitation system (Invitrogen) with specific antibodies, followed by immunoblotting with indicated antibodies as recommended by the supplier. Signal intensity of blotting was determined by densitometry using the NIH-Image J software (http://rsbweb.nih.gov/ij/). Full blot data are shown in Supplementary Fig. 18.
Kanayama M., Inoue M., Danzaki K., Hammer G., He Y.W, & Shinohara M.L. (2015). Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity. Nature communications, 6, 5779.
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2

Immunoprecipitation of A20 and p62 Complexes

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were stimulated with Pam3CSK4 (100 ng/ml) or zymosan (100 μg/ml) and lysed with RIPA buffer and NP-40 (for immunoprecipitation) containing protease inhibitor cocktail (P8340, Sigma). Phosphatase inhibitor cocktail II was added for p-IκB detection. To examine the association between A20 and p62, NIH-3T3 cells were co-transfected with expression vectors for A20 and p62 (pcDNA5-A20-V5/His and pMI-p62-HA, respectively) by using Lipofectamine LTX (Invitrogen), as recommended by the supplier. The expression vectors for A20 and p62 were made in our laboratory, as fusions of the V5/His tag in the pcDNA5 backbone (Invitrogen) and the HA tag in the pMX backbone (Cosmobio), respectively. Immunoprecipitation was carried out by using Dynabeads Protein G Immunoprecipitation system (Invitrogen) with specific antibodies, followed by immunoblotting with indicated antibodies as recommended by the supplier. Signal intensity of blotting was determined by densitometry using the NIH-Image J software (http://rsbweb.nih.gov/ij/). Full blot data are shown in Supplementary Fig. 18.
Kanayama M., Inoue M., Danzaki K., Hammer G., He Y.W, & Shinohara M.L. (2015). Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity. Nature communications, 6, 5779.
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