P52 cflag pcdna3

Manufactured by Addgene

The P52 cFlag pcDNA3 is a plasmid vector that can be used for the expression of proteins with a C-terminal FLAG epitope tag in mammalian cells. The vector contains the CMV promoter for high-level expression and the pcDNA3 backbone.

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Lab products found in correlation

Relb cflag pcdna3, by Addgene (4 mentions) P50 cflag pcdna3, by Addgene (2 mentions) Rela cflag pcdna3, by Addgene (2 mentions) Pcmv myc, by Takara Bio (1 mentions) Pcr flag ikkβ, by Addgene (1 mentions) γ xmg1.2, by Thermo Fisher Scientific (1 mentions) C rel cflag pcdna3, by Addgene (1 mentions) Hsp60 h 1, by Santa Cruz Biotechnology (1 mentions)

Spelling variants of this product

PcDNA3.1 (119 citations)

4 protocols using p52 cflag pcdna3

NF-κB Signaling Pathway Plasmids

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PcDNA-Myc-DBC1 plasmid was purchased from Addgene. Truncation mutants were subcloned into pCMV-Myc (Clontech), and Myc-DBC1-SA mutant was generated by site directed mutagenesis using Advantage GC Rich PCR kit (Clontech) using standard protocol. RelA-cFlag pcDNA3, RelB-cFlag pcDNA3, C-Rel RHD-cFlag-pcDNA3, p50-cFlag-pcDNA3, p52-cFlag pcDNA3, pCR-Flag-IKKα, pCR-Flag-IKKβ plasmids were purchased from Addgene. Antibodies used for immunoblotting and co-immunoprecipitation were anti-p30/DBC1 (Bethyl laboratories), anti-RelB (C-19) (D-4), p100/p52 (C-5), Myc (A-14), (9E10), IKKα/β (H-470) (Santa Cruz), anti-Flag (F7425) (F1804) (Sigma), anti-Tubulin (DM1A) (Calbiochem) anti-phosphoserine (AB1603), phosphothreonine (AB1607) (Millipore).

Kong S., Dong H., Song J., Thiruppathi M., Prabhakar B.S., Qiu Q., Lin Z., Chini E., Zhang B, & Fang D. (2015). Deleted in Breast Cancer 1 (DBC1) suppresses B cell activation through RelB and is regulated by IKKα phosphorylation. Journal of immunology (Baltimore, Md. : 1950), 195(8), 3685-3693.

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Generating NF-κB Signaling Plasmids and Antibodies

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pMIGR1-mP52 and pMIGR1-mGM-CSF were generated by inserting mouse p52 (encoding amino acids 1–405 of NF-κB2) and mouse Csf2 cDNAs, respectively, into the EcoRI and BglII sites of pMIGR1-GFP vector. pcDNA-based expression vectors encoding FLAG-tagged mouse p52 (p52-cFlag-pcDNA3), mouse c-Rel (c-Rel-cFlag-pcDNA3), and mouse RelB (RelB-cFlag-pcDNA3) were from Addgene. The Csf2 promoter luciferase plasmid (pGL3-mCsf2), covering the region −225 to +26 of mouse Csf2 promoter (15 (link)), was provided by Dr. Takeshi Matsumura (Kumamoto University).
Functional grade anti–mouse (m) CD3ɛ (145-2C11) and anti-mCD28 (37.51) antibodies and blocking antibodies for mIFN-γ (XMG1.2) and mIL-4 (11B11) were from eBioscience. Fluorescence-labeled antibodies for mCD4 (L3T4), mCD8 (53-6.7), mCD3 (145-2C11), CD44 (IM7), CD62L (MEL-14), IL-17A (eBio17B7), GM-CSF (MP1-22E9), and IFN-γ (XMG1.2) were purchased from eBioscience. Antibodies for mouse p50 (C-19), c-Rel (sc-70), RelB (C-19), NIK (H-248), Lamin B (C-20), and Hsp60 (H-1) were from Santa Cruz Biotechnology. An antibody recognizing both p100 and p52 (Anti-p52/p100) was provided by NCI Preclinical Repository.

Yu J., Zhou X., Nakaya M., Jin W., Cheng X, & Sun S.C. (2014). T Cell Intrinsic Function of the Noncanonical NF-κB Pathway in the Regulation of GM-CSF Expression and EAE Pathogenesis. Journal of immunology (Baltimore, Md. : 1950), 193(1), 422-430.

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Plasmid Transfection in Primary Cells

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MyD88 flag plasmid was a gift from Ruslan Medzhitov (Addgene plasmid # 13093). RelB cFlag pcDNA3 (Addgene plasmid # 20017) and p52 cFlag pcDNA3 (Addgene plasmid # 20019) were gifts from Stephen Smale. pcDNA3-IKKα-HA WT was a gift from Warner Greene (Addgene plasmid # 23296). Plasmid DNA was electroporated into primary myoblasts (1500 V, 10 ms duration, 3 pulses) or into primary fibroblast (900 V, 40 ms duration, 1 pulse) using the Neon transfection system as previously described^{41 (link), 47 (link)}. This protocol consistently introduced plasmid DNA into ~80% cells.

Hindi S.M., Shin J., Gallot Y.S., Straughn A.R., Simionescu-Bankston A., Hindi L., Xiong G., Friedland R.P, & Kumar A. (2017). MyD88 promotes myoblast fusion in a cell-autonomous manner. Nature Communications, 8, 1624.

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Plasmids for Transcription Factor Studies

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RelA cFlag pcDNA3 (Addgene plasmid # 20012)^{30 (link)}, RelB cFlag pcDNA3 (Addgene plasmid # 20017), p50 cFlag pcDNA3 (Addgene plasmid # 20018) and p52 cFlag pcDNA3 (Addgene plasmid # 20019) were a gift from Stephen Smale. Plasmids expressing both β-galactosidase for transfection control (LacZ pCMV) and luciferase (luc) for luminometry based expression (pLV-5X-NF-κB Luc, pGL3-3X-Ebox Luc, PPRE-3X-TK Luc, pGL2-5X-Gal4 Luc, pGL3basic-Cox2 Promo Luc) were described previously^{31 (link)–35 (link)}. HOPS encoding sequence was amplified and cloned in pCS2+MT and pSG5 as previously described^{1 (link)}. N-terminal myc-tagged plasmids myc-CLOCK-pSG5, Flag-myc-BMAL1 pCS2+MT, Flag-PPARγ1 were previously described^{34 (link)–36 (link)}. pMT123 (HA-(8X)ubiquitin) and pRK5-HA-Ubiquitin (Addgene plasmid # 17608) were gifts from Dirk Bohmann and Ted Dawson respectively. Flag-TRAF6-WT (Addgene plasmid #21624) was a gift from John Kyriakis^{37 (link)}.

Bellet M.M., Pieroni S., Castelli M., Piobbico D., Fallarino F., Romani L., Della-Fazia M.A, & Servillo G. (2020). HOPS/Tmub1 involvement in the NF-kB-mediated inflammatory response through the modulation of TRAF6. Cell Death & Disease, 11(10), 865.

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