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cy5  (Jena Bioscience)
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Jena Bioscience cy5
RNA structure modulates the stoichiometry of synapsin‐1 within condensates. (A) Representative images of in vitro reconstituted EGFP‐synapsin‐1 (Syn 1) and CLN3 condensates with high‐ED and <t>low‐ED</t> <t>Cy5‐labelled</t> CLN3 mutant in the absence of molecular crowder in reaction buffer (150 mM, NaCl, 25 mM Tris‐HCl (pH 7.4), 0.5 mM TCEP). Scale bar, 10 µm. (B) Scatter plot overlayed with Kernel Density Estimate (KDE) shows fluorescence intensities of CLN3 RNA (high‐ED in grey and low‐ED in magenta) as a function of Syn 1 intensities. Each circle represents a single Syn 1/ CLN3 condensate. (C) Top: schematic representation of three‐component in vitro reconstitution system ‐type I (see Methods), with purified EGFP‐synapsin‐1, α‐synuclein‐AF647 (αSyn) and Cy3 labelled CLN3 RNAs. Bottom: Representative micrographs of in vitro reconstituted Syn 1/α‐Syn condensates without or with low‐ED CLN3 RNA. Scale bar, 10 µm. (D) Fluorescence intensity line‐profiles accompanied with corresponding representative images with selected Syn 1/αSyn ± low‐ED CLN3 RNA condensates used to plot the line profiles, where Syn 1 is represented in green, α‐Syn in magenta and low‐ED CLN3 RNA in yellow. (E) Quantifications of Syn 1 and α‐Syn fluorescence intensities expressed as a ratio (Syn 1/αSyn) in reconstituted protein‐protein droplets with or without low‐ or high‐ED CLN3 RNAs. The box plot encompasses the 25 th and 75 th quartiles with the median marked by central line. Whiskers extend to 1.5 times the interquartile range (IQR). Circles indicate outliers. A paired two‐tailed t‐Test comparing values before and after the addition of RNA indicate a p ‐value of <0.00001 and 0.00579 for high‐ED and CLN3 low‐ED respectively. All data were collected from three independent in vitro reconstitutions.
Cy5, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dutp cy5  (Jena Bioscience)
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RNA structure modulates the stoichiometry of synapsin‐1 within condensates. (A) Representative images of in vitro reconstituted EGFP‐synapsin‐1 (Syn 1) and CLN3 condensates with high‐ED and <t>low‐ED</t> <t>Cy5‐labelled</t> CLN3 mutant in the absence of molecular crowder in reaction buffer (150 mM, NaCl, 25 mM Tris‐HCl (pH 7.4), 0.5 mM TCEP). Scale bar, 10 µm. (B) Scatter plot overlayed with Kernel Density Estimate (KDE) shows fluorescence intensities of CLN3 RNA (high‐ED in grey and low‐ED in magenta) as a function of Syn 1 intensities. Each circle represents a single Syn 1/ CLN3 condensate. (C) Top: schematic representation of three‐component in vitro reconstitution system ‐type I (see Methods), with purified EGFP‐synapsin‐1, α‐synuclein‐AF647 (αSyn) and Cy3 labelled CLN3 RNAs. Bottom: Representative micrographs of in vitro reconstituted Syn 1/α‐Syn condensates without or with low‐ED CLN3 RNA. Scale bar, 10 µm. (D) Fluorescence intensity line‐profiles accompanied with corresponding representative images with selected Syn 1/αSyn ± low‐ED CLN3 RNA condensates used to plot the line profiles, where Syn 1 is represented in green, α‐Syn in magenta and low‐ED CLN3 RNA in yellow. (E) Quantifications of Syn 1 and α‐Syn fluorescence intensities expressed as a ratio (Syn 1/αSyn) in reconstituted protein‐protein droplets with or without low‐ or high‐ED CLN3 RNAs. The box plot encompasses the 25 th and 75 th quartiles with the median marked by central line. Whiskers extend to 1.5 times the interquartile range (IQR). Circles indicate outliers. A paired two‐tailed t‐Test comparing values before and after the addition of RNA indicate a p ‐value of <0.00001 and 0.00579 for high‐ED and CLN3 low‐ED respectively. All data were collected from three independent in vitro reconstitutions.
Dutp Cy5, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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aminoallyl dutp cy5  (Jena Bioscience)
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Jena Bioscience aminoallyl dutp cy5
RNA structure modulates the stoichiometry of synapsin‐1 within condensates. (A) Representative images of in vitro reconstituted EGFP‐synapsin‐1 (Syn 1) and CLN3 condensates with high‐ED and <t>low‐ED</t> <t>Cy5‐labelled</t> CLN3 mutant in the absence of molecular crowder in reaction buffer (150 mM, NaCl, 25 mM Tris‐HCl (pH 7.4), 0.5 mM TCEP). Scale bar, 10 µm. (B) Scatter plot overlayed with Kernel Density Estimate (KDE) shows fluorescence intensities of CLN3 RNA (high‐ED in grey and low‐ED in magenta) as a function of Syn 1 intensities. Each circle represents a single Syn 1/ CLN3 condensate. (C) Top: schematic representation of three‐component in vitro reconstitution system ‐type I (see Methods), with purified EGFP‐synapsin‐1, α‐synuclein‐AF647 (αSyn) and Cy3 labelled CLN3 RNAs. Bottom: Representative micrographs of in vitro reconstituted Syn 1/α‐Syn condensates without or with low‐ED CLN3 RNA. Scale bar, 10 µm. (D) Fluorescence intensity line‐profiles accompanied with corresponding representative images with selected Syn 1/αSyn ± low‐ED CLN3 RNA condensates used to plot the line profiles, where Syn 1 is represented in green, α‐Syn in magenta and low‐ED CLN3 RNA in yellow. (E) Quantifications of Syn 1 and α‐Syn fluorescence intensities expressed as a ratio (Syn 1/αSyn) in reconstituted protein‐protein droplets with or without low‐ or high‐ED CLN3 RNAs. The box plot encompasses the 25 th and 75 th quartiles with the median marked by central line. Whiskers extend to 1.5 times the interquartile range (IQR). Circles indicate outliers. A paired two‐tailed t‐Test comparing values before and after the addition of RNA indicate a p ‐value of <0.00001 and 0.00579 for high‐ED and CLN3 low‐ED respectively. All data were collected from three independent in vitro reconstitutions.
Aminoallyl Dutp Cy5, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/cy5+dutp/Aminoallyl-dUTP-Cy5/pm42228570-434-40-41
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cy5 labeling  (Jena Bioscience)
95
Jena Bioscience cy5 labeling
RNA structure modulates the stoichiometry of synapsin‐1 within condensates. (A) Representative images of in vitro reconstituted EGFP‐synapsin‐1 (Syn 1) and CLN3 condensates with high‐ED and <t>low‐ED</t> <t>Cy5‐labelled</t> CLN3 mutant in the absence of molecular crowder in reaction buffer (150 mM, NaCl, 25 mM Tris‐HCl (pH 7.4), 0.5 mM TCEP). Scale bar, 10 µm. (B) Scatter plot overlayed with Kernel Density Estimate (KDE) shows fluorescence intensities of CLN3 RNA (high‐ED in grey and low‐ED in magenta) as a function of Syn 1 intensities. Each circle represents a single Syn 1/ CLN3 condensate. (C) Top: schematic representation of three‐component in vitro reconstitution system ‐type I (see Methods), with purified EGFP‐synapsin‐1, α‐synuclein‐AF647 (αSyn) and Cy3 labelled CLN3 RNAs. Bottom: Representative micrographs of in vitro reconstituted Syn 1/α‐Syn condensates without or with low‐ED CLN3 RNA. Scale bar, 10 µm. (D) Fluorescence intensity line‐profiles accompanied with corresponding representative images with selected Syn 1/αSyn ± low‐ED CLN3 RNA condensates used to plot the line profiles, where Syn 1 is represented in green, α‐Syn in magenta and low‐ED CLN3 RNA in yellow. (E) Quantifications of Syn 1 and α‐Syn fluorescence intensities expressed as a ratio (Syn 1/αSyn) in reconstituted protein‐protein droplets with or without low‐ or high‐ED CLN3 RNAs. The box plot encompasses the 25 th and 75 th quartiles with the median marked by central line. Whiskers extend to 1.5 times the interquartile range (IQR). Circles indicate outliers. A paired two‐tailed t‐Test comparing values before and after the addition of RNA indicate a p ‐value of <0.00001 and 0.00579 for high‐ED and CLN3 low‐ED respectively. All data were collected from three independent in vitro reconstitutions.
Cy5 Labeling, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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500 bp biotin handle  (Jena Bioscience)
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Jena Bioscience 500 bp biotin handle
RNA structure modulates the stoichiometry of synapsin‐1 within condensates. (A) Representative images of in vitro reconstituted EGFP‐synapsin‐1 (Syn 1) and CLN3 condensates with high‐ED and <t>low‐ED</t> <t>Cy5‐labelled</t> CLN3 mutant in the absence of molecular crowder in reaction buffer (150 mM, NaCl, 25 mM Tris‐HCl (pH 7.4), 0.5 mM TCEP). Scale bar, 10 µm. (B) Scatter plot overlayed with Kernel Density Estimate (KDE) shows fluorescence intensities of CLN3 RNA (high‐ED in grey and low‐ED in magenta) as a function of Syn 1 intensities. Each circle represents a single Syn 1/ CLN3 condensate. (C) Top: schematic representation of three‐component in vitro reconstitution system ‐type I (see Methods), with purified EGFP‐synapsin‐1, α‐synuclein‐AF647 (αSyn) and Cy3 labelled CLN3 RNAs. Bottom: Representative micrographs of in vitro reconstituted Syn 1/α‐Syn condensates without or with low‐ED CLN3 RNA. Scale bar, 10 µm. (D) Fluorescence intensity line‐profiles accompanied with corresponding representative images with selected Syn 1/αSyn ± low‐ED CLN3 RNA condensates used to plot the line profiles, where Syn 1 is represented in green, α‐Syn in magenta and low‐ED CLN3 RNA in yellow. (E) Quantifications of Syn 1 and α‐Syn fluorescence intensities expressed as a ratio (Syn 1/αSyn) in reconstituted protein‐protein droplets with or without low‐ or high‐ED CLN3 RNAs. The box plot encompasses the 25 th and 75 th quartiles with the median marked by central line. Whiskers extend to 1.5 times the interquartile range (IQR). Circles indicate outliers. A paired two‐tailed t‐Test comparing values before and after the addition of RNA indicate a p ‐value of <0.00001 and 0.00579 for high‐ED and CLN3 low‐ED respectively. All data were collected from three independent in vitro reconstitutions.
500 Bp Biotin Handle, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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biotin dutp cy5  (Jena Bioscience)
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RNA structure modulates the stoichiometry of synapsin‐1 within condensates. (A) Representative images of in vitro reconstituted EGFP‐synapsin‐1 (Syn 1) and CLN3 condensates with high‐ED and <t>low‐ED</t> <t>Cy5‐labelled</t> CLN3 mutant in the absence of molecular crowder in reaction buffer (150 mM, NaCl, 25 mM Tris‐HCl (pH 7.4), 0.5 mM TCEP). Scale bar, 10 µm. (B) Scatter plot overlayed with Kernel Density Estimate (KDE) shows fluorescence intensities of CLN3 RNA (high‐ED in grey and low‐ED in magenta) as a function of Syn 1 intensities. Each circle represents a single Syn 1/ CLN3 condensate. (C) Top: schematic representation of three‐component in vitro reconstitution system ‐type I (see Methods), with purified EGFP‐synapsin‐1, α‐synuclein‐AF647 (αSyn) and Cy3 labelled CLN3 RNAs. Bottom: Representative micrographs of in vitro reconstituted Syn 1/α‐Syn condensates without or with low‐ED CLN3 RNA. Scale bar, 10 µm. (D) Fluorescence intensity line‐profiles accompanied with corresponding representative images with selected Syn 1/αSyn ± low‐ED CLN3 RNA condensates used to plot the line profiles, where Syn 1 is represented in green, α‐Syn in magenta and low‐ED CLN3 RNA in yellow. (E) Quantifications of Syn 1 and α‐Syn fluorescence intensities expressed as a ratio (Syn 1/αSyn) in reconstituted protein‐protein droplets with or without low‐ or high‐ED CLN3 RNAs. The box plot encompasses the 25 th and 75 th quartiles with the median marked by central line. Whiskers extend to 1.5 times the interquartile range (IQR). Circles indicate outliers. A paired two‐tailed t‐Test comparing values before and after the addition of RNA indicate a p ‐value of <0.00001 and 0.00579 for high‐ED and CLN3 low‐ED respectively. All data were collected from three independent in vitro reconstitutions.
Biotin Dutp Cy5, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cy5 labeled dutp  (Merck & Co)
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Merck & Co cy5 labeled dutp
RNA structure modulates the stoichiometry of synapsin‐1 within condensates. (A) Representative images of in vitro reconstituted EGFP‐synapsin‐1 (Syn 1) and CLN3 condensates with high‐ED and <t>low‐ED</t> <t>Cy5‐labelled</t> CLN3 mutant in the absence of molecular crowder in reaction buffer (150 mM, NaCl, 25 mM Tris‐HCl (pH 7.4), 0.5 mM TCEP). Scale bar, 10 µm. (B) Scatter plot overlayed with Kernel Density Estimate (KDE) shows fluorescence intensities of CLN3 RNA (high‐ED in grey and low‐ED in magenta) as a function of Syn 1 intensities. Each circle represents a single Syn 1/ CLN3 condensate. (C) Top: schematic representation of three‐component in vitro reconstitution system ‐type I (see Methods), with purified EGFP‐synapsin‐1, α‐synuclein‐AF647 (αSyn) and Cy3 labelled CLN3 RNAs. Bottom: Representative micrographs of in vitro reconstituted Syn 1/α‐Syn condensates without or with low‐ED CLN3 RNA. Scale bar, 10 µm. (D) Fluorescence intensity line‐profiles accompanied with corresponding representative images with selected Syn 1/αSyn ± low‐ED CLN3 RNA condensates used to plot the line profiles, where Syn 1 is represented in green, α‐Syn in magenta and low‐ED CLN3 RNA in yellow. (E) Quantifications of Syn 1 and α‐Syn fluorescence intensities expressed as a ratio (Syn 1/αSyn) in reconstituted protein‐protein droplets with or without low‐ or high‐ED CLN3 RNAs. The box plot encompasses the 25 th and 75 th quartiles with the median marked by central line. Whiskers extend to 1.5 times the interquartile range (IQR). Circles indicate outliers. A paired two‐tailed t‐Test comparing values before and after the addition of RNA indicate a p ‐value of <0.00001 and 0.00579 for high‐ED and CLN3 low‐ED respectively. All data were collected from three independent in vitro reconstitutions.
Cy5 Labeled Dutp, supplied by Merck & Co, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


RNA structure modulates the stoichiometry of synapsin‐1 within condensates. (A) Representative images of in vitro reconstituted EGFP‐synapsin‐1 (Syn 1) and CLN3 condensates with high‐ED and low‐ED Cy5‐labelled CLN3 mutant in the absence of molecular crowder in reaction buffer (150 mM, NaCl, 25 mM Tris‐HCl (pH 7.4), 0.5 mM TCEP). Scale bar, 10 µm. (B) Scatter plot overlayed with Kernel Density Estimate (KDE) shows fluorescence intensities of CLN3 RNA (high‐ED in grey and low‐ED in magenta) as a function of Syn 1 intensities. Each circle represents a single Syn 1/ CLN3 condensate. (C) Top: schematic representation of three‐component in vitro reconstitution system ‐type I (see Methods), with purified EGFP‐synapsin‐1, α‐synuclein‐AF647 (αSyn) and Cy3 labelled CLN3 RNAs. Bottom: Representative micrographs of in vitro reconstituted Syn 1/α‐Syn condensates without or with low‐ED CLN3 RNA. Scale bar, 10 µm. (D) Fluorescence intensity line‐profiles accompanied with corresponding representative images with selected Syn 1/αSyn ± low‐ED CLN3 RNA condensates used to plot the line profiles, where Syn 1 is represented in green, α‐Syn in magenta and low‐ED CLN3 RNA in yellow. (E) Quantifications of Syn 1 and α‐Syn fluorescence intensities expressed as a ratio (Syn 1/αSyn) in reconstituted protein‐protein droplets with or without low‐ or high‐ED CLN3 RNAs. The box plot encompasses the 25 th and 75 th quartiles with the median marked by central line. Whiskers extend to 1.5 times the interquartile range (IQR). Circles indicate outliers. A paired two‐tailed t‐Test comparing values before and after the addition of RNA indicate a p ‐value of <0.00001 and 0.00579 for high‐ED and CLN3 low‐ED respectively. All data were collected from three independent in vitro reconstitutions.

Journal: Advanced Science

Article Title: RNA Promotes Synapsin Condensates That Organize Synaptic Vesicles in Live Neurons and Enable Localized Translation in Reconstituted System

doi: 10.1002/advs.77115

Figure Lengend Snippet: RNA structure modulates the stoichiometry of synapsin‐1 within condensates. (A) Representative images of in vitro reconstituted EGFP‐synapsin‐1 (Syn 1) and CLN3 condensates with high‐ED and low‐ED Cy5‐labelled CLN3 mutant in the absence of molecular crowder in reaction buffer (150 mM, NaCl, 25 mM Tris‐HCl (pH 7.4), 0.5 mM TCEP). Scale bar, 10 µm. (B) Scatter plot overlayed with Kernel Density Estimate (KDE) shows fluorescence intensities of CLN3 RNA (high‐ED in grey and low‐ED in magenta) as a function of Syn 1 intensities. Each circle represents a single Syn 1/ CLN3 condensate. (C) Top: schematic representation of three‐component in vitro reconstitution system ‐type I (see Methods), with purified EGFP‐synapsin‐1, α‐synuclein‐AF647 (αSyn) and Cy3 labelled CLN3 RNAs. Bottom: Representative micrographs of in vitro reconstituted Syn 1/α‐Syn condensates without or with low‐ED CLN3 RNA. Scale bar, 10 µm. (D) Fluorescence intensity line‐profiles accompanied with corresponding representative images with selected Syn 1/αSyn ± low‐ED CLN3 RNA condensates used to plot the line profiles, where Syn 1 is represented in green, α‐Syn in magenta and low‐ED CLN3 RNA in yellow. (E) Quantifications of Syn 1 and α‐Syn fluorescence intensities expressed as a ratio (Syn 1/αSyn) in reconstituted protein‐protein droplets with or without low‐ or high‐ED CLN3 RNAs. The box plot encompasses the 25 th and 75 th quartiles with the median marked by central line. Whiskers extend to 1.5 times the interquartile range (IQR). Circles indicate outliers. A paired two‐tailed t‐Test comparing values before and after the addition of RNA indicate a p ‐value of <0.00001 and 0.00579 for high‐ED and CLN3 low‐ED respectively. All data were collected from three independent in vitro reconstitutions.

Article Snippet: Reactions included 1 μg of DNA template and were supplemented with approximately 0.4 μl of Cy3 or Cy5‐labelled UTP (Jena Bioscience, NU‐803‐CY5‐S, NU‐803‐CY3‐S).

Techniques: In Vitro, Mutagenesis, Fluorescence, Purification, Two Tailed Test

Synapsin‐1 condensates promote mRNA translation. (A) Above: images showing Atp5b synaptic mRNA (magenta) enriching synapsin‐1 condensates (Syn 1, green) in in vitro system under physiological conditions and in the absence of any molecular crowders. Scale bar, 5 µm. Below: Scatter plot depicts quantified Atp5b RNA intensities as a function of Syn 1 intensities. Each circle represents individual condensate analyzed overlaying Kernel Density Estimate (KDE). Data were collected from three independent in vitro reconstitutions of in vitro transcribed Cy5 labelled Atp5b and purified EGFP‐tagged synapsin‐1. (B) Top: scheme represents simplified overview of the in vitro luciferase translation assay using Atp5b ‐nano‐luciferase ( Atp5b::Nluc ) as an RNA reporter. Below: representative images of Syn 1 and Syn 1/α‐Syn condensates in rabbit reticulocyte extract (RR). Scale bar, 5 µm. (C) The violin plot showing the quantification of the Atp5b::Nluc luminescence signal under different reaction conditions (only Atp5b::Nluc, Atp5b::Nluc + BSA , Atp5b::Nluc + Syn 1, Atp5b::Nluc + Syn 1 + αSyn, and Atp5b::Nluc + Syn 1RtoK). Within each violin plot, the box depicts the 25th through the 75th quartiles, with whiskers extending to min and max data point. Each individual circle represents a technical replicate. Data from from 3 independent extracts. Colored black circles below the plot indicate component present in the reaction mix. (D) Scheme of MoonTag system developed to quantitatively detect translation events live in cellulo and in our work optimized for in vitro system of Syn 1 condensates in RR extract. (E) Top: Scheme of the MoonTag translation pipeline in RR extract using Atp5b‐ MoonTag ( Atp5b::Moon ) RNA reporter system. Bottom: Representative images of Syn 1 (green) and MoonTag(‐Nb) nanobody signal (magenta) in condensates enriched with Syn 1 and Atp5b :Nluc used as negative control (upper image panel) and Atp5b::MoonTag reporter RNA (lower image panel) 60 min after the addition of RR to pre‐formed synapsin‐1/RNA condensates. Scale bar, 2 µm. (F) Box plot showing anti‐MoonTag nanobody (2H10Nb) fluorescence values for the different in vitro conditions (only Atp5b::Nluc+ Synapsin‐1 [negative control], Atp5b::Moon + Syn 1 , Atp5b::Moon + Syn 1+ αSyn) 60 min after the addition of RR. For each box plot, the horizontal line in the box plot denotes the median value of data distribution between 25 th and 75 th percentiles. Whiskers extend to 1.5× the IQR. Each circle represents individual condensate analyzed. Data were collected from 3 independent RR extracts. Paired, two‐tailed t‐tests were carried out comparing mCherry‐2H10Nb signal values between reactions containing Atp5b::Nluc and two conditions: (1) Atp5b::Moon +Syn 1 and (2) Atp5b::Moon +Syn 1+ αSyn; P‐values <0.0001 (****), for both conditions respectively. First comparison is highlighted on the graph (****). The same statistical test is performed to compare Atp5b::Moon +Syn 1 and Atp5b::Moon +Syn 1+αSyn conditions ( p ‐value = 0.001166). (G) Box plot showing the fraction of pixels within each condensate whose Syn 1 fluorescence signal is less than 0.8 × Median Syn1 pixel value of the condensate. This value represents the mask of the segmented ‘translation‐hubs’. Values are displayed for condensates in RR treated without or with Cycloheximide (CHX). For each box plot, the horizontal line in the box plot denotes the median value of data distribution between 25th and 75th percentiles. Whiskers extend to 1.5× the IQR. Each circle represents individual condensate analyzed. Data were collected from 3 independent RR extracts. A Welch's T‐test was used to compare the fraction of dim pixels per condensate between CHX treated and non‐treated RRs. p ‐value = 0.0001767 (**).

Journal: Advanced Science

Article Title: RNA Promotes Synapsin Condensates That Organize Synaptic Vesicles in Live Neurons and Enable Localized Translation in Reconstituted System

doi: 10.1002/advs.77115

Figure Lengend Snippet: Synapsin‐1 condensates promote mRNA translation. (A) Above: images showing Atp5b synaptic mRNA (magenta) enriching synapsin‐1 condensates (Syn 1, green) in in vitro system under physiological conditions and in the absence of any molecular crowders. Scale bar, 5 µm. Below: Scatter plot depicts quantified Atp5b RNA intensities as a function of Syn 1 intensities. Each circle represents individual condensate analyzed overlaying Kernel Density Estimate (KDE). Data were collected from three independent in vitro reconstitutions of in vitro transcribed Cy5 labelled Atp5b and purified EGFP‐tagged synapsin‐1. (B) Top: scheme represents simplified overview of the in vitro luciferase translation assay using Atp5b ‐nano‐luciferase ( Atp5b::Nluc ) as an RNA reporter. Below: representative images of Syn 1 and Syn 1/α‐Syn condensates in rabbit reticulocyte extract (RR). Scale bar, 5 µm. (C) The violin plot showing the quantification of the Atp5b::Nluc luminescence signal under different reaction conditions (only Atp5b::Nluc, Atp5b::Nluc + BSA , Atp5b::Nluc + Syn 1, Atp5b::Nluc + Syn 1 + αSyn, and Atp5b::Nluc + Syn 1RtoK). Within each violin plot, the box depicts the 25th through the 75th quartiles, with whiskers extending to min and max data point. Each individual circle represents a technical replicate. Data from from 3 independent extracts. Colored black circles below the plot indicate component present in the reaction mix. (D) Scheme of MoonTag system developed to quantitatively detect translation events live in cellulo and in our work optimized for in vitro system of Syn 1 condensates in RR extract. (E) Top: Scheme of the MoonTag translation pipeline in RR extract using Atp5b‐ MoonTag ( Atp5b::Moon ) RNA reporter system. Bottom: Representative images of Syn 1 (green) and MoonTag(‐Nb) nanobody signal (magenta) in condensates enriched with Syn 1 and Atp5b :Nluc used as negative control (upper image panel) and Atp5b::MoonTag reporter RNA (lower image panel) 60 min after the addition of RR to pre‐formed synapsin‐1/RNA condensates. Scale bar, 2 µm. (F) Box plot showing anti‐MoonTag nanobody (2H10Nb) fluorescence values for the different in vitro conditions (only Atp5b::Nluc+ Synapsin‐1 [negative control], Atp5b::Moon + Syn 1 , Atp5b::Moon + Syn 1+ αSyn) 60 min after the addition of RR. For each box plot, the horizontal line in the box plot denotes the median value of data distribution between 25 th and 75 th percentiles. Whiskers extend to 1.5× the IQR. Each circle represents individual condensate analyzed. Data were collected from 3 independent RR extracts. Paired, two‐tailed t‐tests were carried out comparing mCherry‐2H10Nb signal values between reactions containing Atp5b::Nluc and two conditions: (1) Atp5b::Moon +Syn 1 and (2) Atp5b::Moon +Syn 1+ αSyn; P‐values <0.0001 (****), for both conditions respectively. First comparison is highlighted on the graph (****). The same statistical test is performed to compare Atp5b::Moon +Syn 1 and Atp5b::Moon +Syn 1+αSyn conditions ( p ‐value = 0.001166). (G) Box plot showing the fraction of pixels within each condensate whose Syn 1 fluorescence signal is less than 0.8 × Median Syn1 pixel value of the condensate. This value represents the mask of the segmented ‘translation‐hubs’. Values are displayed for condensates in RR treated without or with Cycloheximide (CHX). For each box plot, the horizontal line in the box plot denotes the median value of data distribution between 25th and 75th percentiles. Whiskers extend to 1.5× the IQR. Each circle represents individual condensate analyzed. Data were collected from 3 independent RR extracts. A Welch's T‐test was used to compare the fraction of dim pixels per condensate between CHX treated and non‐treated RRs. p ‐value = 0.0001767 (**).

Article Snippet: Reactions included 1 μg of DNA template and were supplemented with approximately 0.4 μl of Cy3 or Cy5‐labelled UTP (Jena Bioscience, NU‐803‐CY5‐S, NU‐803‐CY3‐S).

Techniques: In Vitro, Purification, Luciferase, Negative Control, Fluorescence, Two Tailed Test, Comparison