rfp eea1 (Addgene inc)

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Addgene inc rfp eea1

( A ) Schematic representation of the experimental design used to measure luminescence upon proximity between Rap1GAP-SmBiT and LgBiT-FYVE in response to CCR7 activation. ( B ) Change in luminescence measured upon stimulation of HEK293-CCR7 cells expressing Rap1GAP-SmBiT and LgBiT-FYVE in response to 100 nM CCL19 or 100 nM CCL21 stimulation. The response to chemokine stimulation was normalized to vehicle control. ( C ) Area under the curve (AUC) was used to calculate the total response for each chemokine ligand. Data represent the mean ± SE of N=4 experiments. ( D ) Schematic representation of the enhanced bystander bioluminescence resonance energy transfer (EbBRET)-based assay to monitor proximity between RlucII-miniGi and the endosomal marker rGFP-Rab5 upon CCR7 activation from endosomes. ( E ) EbBRET measurements from CCR7-expressing HEK293 cells co-transfected with RlucII-miniGi and rGFP-Rab5 upon stimulation with 100 nM CCL19, 100 nM CCL21, or vehicle control. Data represents the mean ± SE from N=5 independent experiments. ( F ) Confocal microscopy imaging displaying CCR7-expressing HEK293 cells co-transfected with the plasma membrane marker RFP-Lck and Halo-miniGi. The cells were stimulated with 100 nM CCL19, 100 nM CCL21, or vehicle control for 10 min. Representative images from N=3 experiments. ( G ) Co-localization quantification analysis of RFP-Lck and Halo-miniGi from six representative confocal microscopy images per condition. ( H ) Confocal microscopy imaging displaying CCR7-expressing HEK293 cells co-transfected with the endosomal marker <t>RFP-EEA1</t> and Halo-miniGi. The cells were stimulated with 100 nM CCL19, 100 nM CCL21, or vehicle control for 30 min. Representative images from N=3 experiments. ( I ) Co-localization quantification analysis of RFP-EEA1 and Halo-miniGi from five confocal microscopy images per condition. ( C, E, G, and I ) One-way ANOVA with Tukey’s multiple comparison post hoc tests were applied to determine statistical differences between the distinct treatments (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Rfp Eea1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pegfpc1+expression+vector/TagRFP-T-EEA1+(Plasmid+%2342635)/pmc11850004-253-0-21
Average 93 stars, based on 27 article reviews

rfp eea1 - by Bioz Stars, 2026-09

93/100 stars

Images

1) Product Images from "Endosomal chemokine receptor signalosomes regulate central mechanisms underlying cell migration"

Article Title: Endosomal chemokine receptor signalosomes regulate central mechanisms underlying cell migration

Journal: eLife

doi: 10.7554/eLife.99373

Figure Legend Snippet: ( A ) Schematic representation of the experimental design used to measure luminescence upon proximity between Rap1GAP-SmBiT and LgBiT-FYVE in response to CCR7 activation. ( B ) Change in luminescence measured upon stimulation of HEK293-CCR7 cells expressing Rap1GAP-SmBiT and LgBiT-FYVE in response to 100 nM CCL19 or 100 nM CCL21 stimulation. The response to chemokine stimulation was normalized to vehicle control. ( C ) Area under the curve (AUC) was used to calculate the total response for each chemokine ligand. Data represent the mean ± SE of N=4 experiments. ( D ) Schematic representation of the enhanced bystander bioluminescence resonance energy transfer (EbBRET)-based assay to monitor proximity between RlucII-miniGi and the endosomal marker rGFP-Rab5 upon CCR7 activation from endosomes. ( E ) EbBRET measurements from CCR7-expressing HEK293 cells co-transfected with RlucII-miniGi and rGFP-Rab5 upon stimulation with 100 nM CCL19, 100 nM CCL21, or vehicle control. Data represents the mean ± SE from N=5 independent experiments. ( F ) Confocal microscopy imaging displaying CCR7-expressing HEK293 cells co-transfected with the plasma membrane marker RFP-Lck and Halo-miniGi. The cells were stimulated with 100 nM CCL19, 100 nM CCL21, or vehicle control for 10 min. Representative images from N=3 experiments. ( G ) Co-localization quantification analysis of RFP-Lck and Halo-miniGi from six representative confocal microscopy images per condition. ( H ) Confocal microscopy imaging displaying CCR7-expressing HEK293 cells co-transfected with the endosomal marker RFP-EEA1 and Halo-miniGi. The cells were stimulated with 100 nM CCL19, 100 nM CCL21, or vehicle control for 30 min. Representative images from N=3 experiments. ( I ) Co-localization quantification analysis of RFP-EEA1 and Halo-miniGi from five confocal microscopy images per condition. ( C, E, G, and I ) One-way ANOVA with Tukey’s multiple comparison post hoc tests were applied to determine statistical differences between the distinct treatments (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Techniques Used: Activation Assay, Expressing, Control, Bioluminescence Resonance Energy Transfer, Marker, Transfection, Confocal Microscopy, Imaging, Membrane, Comparison

Amplification:

Article Title: Target-locking single-molecule nanoscopy
Article Snippet: TetR was amplified from pCDNA6/TR (Life Technologies) and cloned into pCDNA4/TO. .. TagRFP was amplified from TagRFP-T-EEA1 (Addgene 42635) with a reverse primer containing an SV40 Nuclear-localization signal (NLS) sequence and cloned into pCDNA4/TO-tetR, resulting in tetR-RFP. .. Alternatively, the fluorescence was separated using a quad-view device (Photometrics, QV2) and GFP, RFP and SiR were simultaneously imaged onto different quadrants of a back-illuminated EM-CCD detector (Andor iXon3 897).

Sequencing:

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism
Article Snippet: After blunting with T4 DNA polymerase (Thermo Fisher Scientific), the remainder was self-ligated. .. For construction of plasmids containing TagRFP-T-Mieap (pTagRFP-T-Mieap), the nucleotide sequence of pEGFP-Mieap between the Nhe I and Xho I restriction sites containing EGFP was replaced with nucleotide sequence of pTagRFP-T-EEA1 (Addgene #42635) between the Nhe I and Xho I restriction sites containing TagRFP-T, by digestion using Nhe I and Xho I. .. For construction of plasmids containing GST-Mieap (pGST-Mieap), the nucleotide sequence of Mieap (amino acids 99–298) was PCR-amplified using the primers, GST-Mieap-F and GST-Mieap-R. PCR products were ligated into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific) and sequenced.

Clone Assay:

other:

Article Title: Endosomal chemokine receptor signalosomes regulate central mechanisms underlying cell migration
Article Snippet: Coding sequence for Rac1, PAK1- GDB, Cdc42, and WAS- GBD were human codon- optimized and gene- synthesized by Genscript and inserted into the pCAGGS plasmid using a NEBuilder assembly kit.

Article Title: RABEP1 amplifies front signaling in neutrophil migration
Article Snippet: PrimeSTAR MAX DNA Polymerase (R045), In-Fusion Snap Assembly Master Mix (ST2320), SOC media (ST0215), Lenti-X concentrator (631232) and Stellar Competent Cells (636763) were from Takara Bio (San Jose, CA, USA).

Construct:

Article Title: Endosomal chemokine receptor signalosomes regulate central mechanisms underlying cell migration
Article Snippet: Halo-miniGi (also referred to as miniGsi) was kindly provided by Prof. Nevin A. Lambert (Augusta University, USA). .. RFP-EEA1 (TagRFP-T-EEA1 cloned into pEGFP-C1 vector) and HA-Dyn-K44A (cloned into pcDNA3.1) constructs were gifts from Silvia Corvera and Sandra Schmid (respectively Addgene plasmids #42635 and #34683). .. RFP-Lck (C-tRFP-Lck cloned into PCMV6-AC-RFP expression vector) was purchased from Origene (#RC100049).

Plasmid Preparation:

Article Title: Plasma membrane rather than endosomal Gq signaling drives transcriptional activity by the viral chemokine receptor US28 in glioblastoma
Article Snippet: C-tRFP-Lck cloned into PCMV6-AC-RFP expression vector was purchased from Origene (#RC100049). .. TagRFP-T-EEA1 in pEGFP-C1 vector and K44A HA-dynamin 1 in pcDNA3.1 were respective gifts from Silvia Corvera and Sandra Schmid (Addgene plasmids #42635 and #34683). β-arrestin1-GFP10 and rGFP-Rab7 in pcDNA3.1+ was a kind gift from Prof. Michel Bouvier (Université de Montréal, Canada) and Stéphane Laporte (McGill University, Canada). .. Renilla reniformis luciferase II (Rluc), US28-Rluc, and US28-PDT-Rluc in pcDNA3.1+ were synthetized by GenScript.

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