Pegfp n3

Manufactured by Thermo Fisher Scientific

Sourced in United States

PEGFP-N3 is a mammalian expression vector that encodes a red-shifted variant of Aequorea victoria green fluorescent protein (GFP). It is designed for monitoring gene expression and protein localization studies in living cells.

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Lab products found in correlation

Anti β tubulin, by Abcam (1 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions) Fast mutagenesis kit v2, by Vazyme (1 mentions) Pitstop 2, by Merck Group (1 mentions) Pcdna3.1 flag, by Thermo Fisher Scientific (1 mentions) Peroxidase conjugated affinipure goat anti rabbit igg, by Proteintech (1 mentions) Alexa fluor 647, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Merck Group (1 mentions) Quickchange mutagenesis kit, by Agilent Technologies (1 mentions) Pcdna3.1 ha, by Thermo Fisher Scientific (1 mentions) Recombinant ampk α β γ proteins, by Merck Group (1 mentions) Pgl3 luciferase reporter plasmid, by Promega (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)

3 protocols using pegfp n3

Clathrin Light Chain Mutant Construction

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Hoechst 33342 and Pitstop 2 were purchased from Sigma-Aldrich. Pitstop 2 were dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions. The lipophilic dyes DiO and DiD were purchased from Biotium. The fluorescent dyes Alexa Fluor 647 and Alexa Fluor 488 phalloidin were purchased from Invitrogen. anti-β-tubulin was purchased from Abcam (USA). peroxidase-conjugated affinipure goat anti-rabbit IgG were purchased from proteintech (USA).
Using the primers listed in Table 1, the full-length CLCs were constructed in vectors including pcDNA3.1-flag, pEGFP-N3, and pmDsRed-C1 (Invitrogen). Site-directed mutants, including EaCLCa-W119R and EaCLCb-W122R, were all subcloned into the pEGFP-N3, pmDsRed-C1 and pcDNA3.1-flag vectors using specific primers (Table 1) and the Fast Mutagenesis Kit V2 (Vazyme). Tryptophan (W)119 of CLCa and W122 of CLCb were both replaced with arginine(R). In addition, the pEGFP-Rab5 vector was maintained in our laboratory. The constructed plasmids were confirmed by sequencing.

Wang L., Li Q., Ni S., Huang Y., Wei J., Liu J., Yu Y., Wang S, & Qin Q. (2019). The roles of grouper clathrin light chains in regulating the infection of a novel marine DNA virus, Singapore grouper iridovirus. Scientific Reports, 9, 15647.

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AMPK-α2 Regulation by Src and FAK

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Homo Sapiens AMPK-α2 cDNA was a gift from Stefan Riedl (Sanford Burnham Prebys Medical Discovery Institute, originally from Dharmacon, Lafayette, CO), and was constructed into pcDNA3.1-HA and pEGFP-N3 (Invitrogen) vectors to express HA- or GFP-tagged AMPK-α2. AMPK-β2-Flag (Addgene plasmid # 40603) and AMPK-γ1-HA (Addgene plasmid # 40605) (26 ) were gifts from Harvey Lodish (MIT, Cambridge, MA). Wild-type (WT) Src, kinase-dead (KD) Src (K295N), active Src (Y527F), HA-FAK and various FAK mutants have been described (27 (link)). Phospho-mimetic mutant AMPK-α2-Y179E and phosphorylation-resistant mutant AMPK-α2-Y179F were created by using the Quick Change Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Recombinant AMPK-α/β/γ proteins were from EMD Millipore; recombinant GST-tagged active Src kinase was from (R&D Systems Inc., Minneapolis, MN).

Zhao M., Finlay D., Kwong E., Liddington R., Viollet B., Sasaoka N, & Vuori K. (2021). Cell adhesion suppresses autophagy via Src/FAK-mediated phosphorylation and inhibition of AMPK. Cellular signalling, 89, 110170.

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Overexpression of CCN2 and AKT3 via miR-610

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For ectopic expression of CCN2 and AKT3, CCN2 open reading frames (ORFs) and AKT3 ORFs with a 3′-untranslated region (UTR) were amplified using polymerase chain reaction (PCR) and subcloned into pEGFP-N3 (Invitrogen Life Technologies). To construct a luciferase reporter vector, the CCN2 and AKT3 3′-UTR fragment containing putative binding sites for miR-610 were amplified using PCR and cloned downstream of the luciferase gene in the pGL3-luciferase reporter plasmid (Promega Corporation, Madison, WI, USA). The miR-610 mimics, negative control, and miR-610 inhibitor were purchased from Genecopoeia (Rockville, MD, USA). The transfections were performed with Lipofectamine 2000 reagent (Invitrogen Life Technologies).

MO X., CAO Q., LIANG H., LIU J., LI H, & LIU F. (2016). MicroRNA-610 suppresses the proliferation of human glioblastoma cells by repressing CCND2 and AKT3. Molecular Medicine Reports, 13(3), 1961-1966.

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