mrna concentration  (Thermo Fisher)

Journal: The Journal of Biological Chemistry

Article Title: 4E-BP1 differentially regulates translation of a subset of human mRNAs

doi: 10.1016/j.jbc.2025.110976

Figure Lengend Snippet: Fluorescence anisotropy analyses of eIF4E and eIF4E•4E-BP1 binding to m 7 G-capped 5′UTRs of HIF-1α, p53 A , FGF-9, and p53 B encoding mRNAs . Normalized anisotropy changes (denoted as r̄) for the interaction of eIF4E (-▪-), eIF4E•4E-BP1 ( ), and 4E-BP1 ( ) with 10 nM m 7 G-capped 5′UTR of ( A ) β-Actin (control), ( B ) HIF-1α, ( C ) p53 A , ( D ) FGF-9 and ( E ) p53 B encoding mRNA transcripts fluorescein-labeled at 3′terminus, respectively. Data points corresponding to the average from three independent anisotropy measurements were normalized and plotted against protein concentration (μM). Curves represent Hill-equation fits, as described in materials and methods, used to calculate the corresponding K D values.

Article Snippet: Ethanol precipitation followed using the mRNA Clean and Concentrator Kit from Zymo Research following the manufacturer’s protocol ( ).

Techniques: Fluorescence, Binding Assay, Control, Labeling, Protein Concentration

Journal: The Journal of Biological Chemistry

Article Title: 4E-BP1 differentially regulates translation of a subset of human mRNAs

doi: 10.1016/j.jbc.2025.110976

Figure Lengend Snippet: Fluorescence anisotropy analyses of eIF4E and eIF4E•4E-BP1 binding to ApppG-capped 5′UTRs of HIF-1α, p53 A , FGF-9, and p53 B encoding mRNAs . Normalized anisotropy changes (denoted as r̄) for the interaction of eIF4E (-▪-), eIF4E•4E-BP1 ( ), and 4E-BP1 ( ) with 10 nM ApppG-capped 5′UTR of ( A ) HIF-1α, ( B ) p53 A , ( C ) FGF-9, and ( D ) p53 B encoding mRNA transcripts fluorescein-labeled at 3′terminus, respectively. Data points corresponding to the average from three independent anisotropy measurements were normalized and plotted against protein concentration (μM). Curves represent Hill-equation fits, as described in materials and methods, used to calculate the corresponding K D values.

Article Snippet: Ethanol precipitation followed using the mRNA Clean and Concentrator Kit from Zymo Research following the manufacturer’s protocol ( ).

Techniques: Fluorescence, Binding Assay, Labeling, Protein Concentration

Journal: The Journal of Biological Chemistry

Article Title: 4E-BP1 differentially regulates translation of a subset of human mRNAs

doi: 10.1016/j.jbc.2025.110976

Figure Lengend Snippet: 4E-BP1 effects on the translation yields of m 7 G- and ApppG-capped-5′UTR-Luc mRNAs . A , Cartoon representation of the mRNA reporter used for this study. Translation yields of ( B ) m 7 G-β-actin-UTR-Luc mRNA; Comparison of m 7 G- and ApppG-capped transcripts of ( C ) HIF-1α; ( D ) p53 A , ( E ) FGF-9, and ( F ) p53 B -UTR-Luc encoding mRNAs following addition of increasing concentrations of 4E-BP1. Relative luciferase activity was normalized to the respective controls (m 7 G-capped-UTR-Luc mRNA) for each reporter constructs with no 4E-BP1 added to non-depleted RRL. Diagonally striped bar (- -), represents m 7 G-capped mRNAs; and white bar (- -), represents ApppG-capped RNAs, respectively. Bar heights and error bars correspond to the average and standard deviations, respectively, of three independent luciferase activity measurements. Data were analyzed by two-tailed unpaired Student’s t test, where p represents the probability that differences occurred by chance: n.s, p = 0.12; ∗, p < 0.033; ∗∗, p = 0.002; ∗∗∗, p < 0.001.

Article Snippet: Ethanol precipitation followed using the mRNA Clean and Concentrator Kit from Zymo Research following the manufacturer’s protocol ( ).

Techniques: Comparison, Luciferase, Activity Assay, Construct, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: 4E-BP1 differentially regulates translation of a subset of human mRNAs

doi: 10.1016/j.jbc.2025.110976

Figure Lengend Snippet: Effect of eIF4E on the translation yields of m 7 G- and ApppG-capped HIF-1α, p53 A , FGF-9, and p53 B -5′UTR-Luc mRNA transcripts . Translation yields of m 7 G-capped ( A – D ) and ApppG-capped ( E – H ) mRNA transcripts of HIF-1α-, p53 A -, FGF-9-, and p53 B -UTR-Luc mRNAs, respectively, following treatment of the RRL with 50 nM of 4E-BP1 and increasing concentrations of eIF4E. Conditions included 50 nM (denoted as +), and 150 nM (denoted as ++) of eIF4E. For m 7 G-capped mRNAs, 4E-BP1 alone ( diagonally striped bar , - -); increasing eIF4E concentration ( vertically striped bar , - -). For ApppG-capped mRNAs, 4E-BP1 alone ( white bar , - -); increasing eIF4E concentration ( vertically striped bar , - -).4E-BP1 was held constant at 50 nM in all conditions. Relative luciferase activity was normalized to the respective controls (ApppG-capped-UTR-Luc mRNA) for each reporter constructs with no 4E-BP1 added to RRL. Bar heights and error bars correspond to the average and standard deviations, respectively, of three independent luciferase activity measurements. Data were analyzed by two-tailed unpaired Student’s t test, where p represents the probability that differences occurred by chance: n.s, p = 0.12; ∗, p < 0.033; ∗∗, p = 0.002; ∗∗∗, p < 0.001.

Article Snippet: Ethanol precipitation followed using the mRNA Clean and Concentrator Kit from Zymo Research following the manufacturer’s protocol ( ).

Techniques: Concentration Assay, Luciferase, Activity Assay, Construct, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: 4E-BP1 differentially regulates translation of a subset of human mRNAs

doi: 10.1016/j.jbc.2025.110976

Figure Lengend Snippet: Effect of eIF4GI 557-1599 • eIF4A on the translation yields of m 7 G- and ApppG-capped HIF-1α, p53 A , FGF-9, and p53 B -5′UTR-Luc mRNA transcripts . Translation yields of m 7 G-capped ( A – D ) and ApppG-capped ( E – H ) mRNA transcripts of HIF-1α-, p53 A -, FGF-9-, and p53 B -UTR-Luc mRNAs, respectively, following treatment of the RRL with 50 nM of 4E-BP1 and increasing concentrations of eIF4GI 557-1599 •eIF4A (added at a 1:1 M ratio). Conditions included 50 nM (denoted as +), and 150 nM (denoted as ++) of eIF4GI 557-1599 •eIF4A. For m 7 G-capped mRNAs, 4E-BP1 alone ( diagonally striped bar , - -); increasing eIF4GI•eIF4A concentration ( horizontally striped bar , - -). For ApppG-capped mRNAs, 4E-BP1 alone ( white bar , - -); increasing eIF4GI•eIF4A concentration ( horizontally striped bar , - -). 4E-BP1 was held constant at 50 nM in all conditions. Relative luciferase activity was normalized to the respective controls (m 7 G-capped-UTR-Luc mRNA) for each reporter constructs with no 4E-BP1 added to RRL. Bar heights and error bars correspond to the average and standard deviations, respectively, of three independent luciferase activity measurements. Data were analyzed by two-tailed unpaired Student’s t test, where p represents the probability that differences occurred by chance: n.s, p = 0.12; ∗, p < 0.033; ∗∗, p = 0.002; ∗∗∗, p < 0.001.

Article Snippet: Ethanol precipitation followed using the mRNA Clean and Concentrator Kit from Zymo Research following the manufacturer’s protocol ( ).

Techniques: Concentration Assay, Luciferase, Activity Assay, Construct, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: 4E-BP1 differentially regulates translation of a subset of human mRNAs

doi: 10.1016/j.jbc.2025.110976

Figure Lengend Snippet: Fluorescence anisotropy analyses of eIF4GI 557-1599 (alone or in complex with eIF4A ± ATP) binding to m 7 G-capped 5′UTRs of HIF-1α, p53 A , FGF-9, and p53 B encoding mRNAs either alone or as part of the mRNA • eIF4E • 4E-BP1 ternary complex . Normalized anisotropy changes (denoted as r̄) for the interaction of eIF4GI 557-1599 binding to mRNAs alone (-▪-) and mRNA•eIF4E•4E-BP1 ( ); eIF4GI 557-1599 •eIF4A binding to mRNA•eIF4E•4E-BP1 ( ), and eIF4GI 557-1599 •eIF4A•ATP to mRNA•eIF4E•4E-BP1 ( ) with 10 nM m 7 G-capped 5′UTR of ( A ) HIF-1α, ( B ) p53 A , ( C ) FGF-9 and ( D ) p53 B encoding mRNA transcripts fluorescein-labeled at 3′terminus, respectively. Data points corresponding to the average from three independent anisotropy measurements were normalized and plotted against protein concentration (μ M). Curves represent Hill-equation fits, as described in materials and methods, used to calculate the corresponding K D values.

Article Snippet: Ethanol precipitation followed using the mRNA Clean and Concentrator Kit from Zymo Research following the manufacturer’s protocol ( ).

Techniques: Fluorescence, Binding Assay, Labeling, Protein Concentration

Journal: The Journal of Biological Chemistry

Article Title: 4E-BP1 differentially regulates translation of a subset of human mRNAs

doi: 10.1016/j.jbc.2025.110976

Figure Lengend Snippet: Fluorescence anisotropy analyses of eIF4GI 557-1599 (alone or in complex with eIF4A ± ATP) binding to ApppG-capped 5′UTRs of HIF-1α, p53 A , FGF-9, and p53 B encoding mRNAs either alone or as part of the mRNA • eIF4E • 4E-BP1 ternary complex . Normalized anisotropy changes (denoted as r̄) for the interaction of eIF4GI 557-1599 binding to mRNAs alone (-▪-) and mRNA•eIF4E•4E-BP1 ( ); eIF4GI 557-1599 •eIF4A binding to mRNA•eIF4E•4E-BP1 ( ), and eIF4GI 557-1599 •eIF4A•ATP to mRNA•eIF4E•4E-BP1 ( ) with 10 nM ApppG-capped 5′UTR of ( A ) HIF-1α, ( B ) p53 A , ( C ) FGF-9 and ( D ) p53 B encoding mRNA transcripts fluorescein-labeled at 3′terminus, respectively. Data points corresponding to the average from three independent anisotropy measurements were normalized and plotted against protein concentration (μ M). Curves represent Hill-equation fits, as described in materials and methods, used to calculate the corresponding K D values.

Article Snippet: Ethanol precipitation followed using the mRNA Clean and Concentrator Kit from Zymo Research following the manufacturer’s protocol ( ).

Techniques: Fluorescence, Binding Assay, Labeling, Protein Concentration

Journal: The Journal of Biological Chemistry

Article Title: 4E-BP1 differentially regulates translation of a subset of human mRNAs

doi: 10.1016/j.jbc.2025.110976

Figure Lengend Snippet: Fluorescence anisotropy analyses of eIF4GI 557-1599 binding to mRNA•eIF4E•4E-BP1-FITC-Peptide ternary complex . Normalized anisotropy changes (denoted as r̄) for the interaction of eIF4GI 557-1599 binding to pre-assembled mRNA•eIF4E•4E-BP1-FITC-peptide ternary complexes in the presence of different 5′capped mRNAs. Each mRNA was tested in both m 7 G-capped ( A ) and ApppG-capped ( B ) forms. 5′UTRs of mRNA encoding HIF-1α (-▪-), p53 A ( ), FGF-9 ( ), and p53 B ( ) were analyzed. All assays were performed using 20 nM fluorescein-labeled 4E-BP1 peptide. Data points corresponding to the average from three independent anisotropy measurements were normalized and plotted as a function of eIF4GI 557-1599 concentration (μ M). Curves represent Hill-equation fits, as deciphered in materials and methods, used to calculate the corresponding K D values.

Article Snippet: Ethanol precipitation followed using the mRNA Clean and Concentrator Kit from Zymo Research following the manufacturer’s protocol ( ).

Techniques: Fluorescence, Binding Assay, Labeling, Concentration Assay

Journal: bioRxiv

Article Title: Circular mRNA against CleanCap linear mRNA vectors: comprehensive comparison, expression, active and passive immunization

doi: 10.1101/2025.10.13.682008

Figure Lengend Snippet: schematic process of mRNA-vectors production. Linear vectors contained either uridine or N1-methylpseudouridine and were capped with ARCA reagent or CleanCap AG reagents. Circular vectors include either CVB3 or HRVB6 IRES and did not contain N1-methylpseudouridine.

Article Snippet: Final IVT products were purified with the mRNA Clean & Concentrator Kit (ZYMO #R1018).

Techniques:

Journal: bioRxiv

Article Title: Circular mRNA against CleanCap linear mRNA vectors: comprehensive comparison, expression, active and passive immunization

doi: 10.1101/2025.10.13.682008

Figure Lengend Snippet: In vitro kinetics of luciferase activity at HEK-293 cell line. (The experiment was performed three times at different cell passages; for each experiment all the mRNA-vectors were freshly prepared.

Article Snippet: Final IVT products were purified with the mRNA Clean & Concentrator Kit (ZYMO #R1018).

Techniques: In Vitro, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Circular mRNA against CleanCap linear mRNA vectors: comprehensive comparison, expression, active and passive immunization

doi: 10.1101/2025.10.13.682008

Figure Lengend Snippet: SARS-CoV2 experiment. (A) – Immunogenicity of circular and linear mRNA vectors after prime-boost vaccination at a dose 3 µg. (B) – Viral load measured by PCR-RT in lungs of immunized mice on day 3 post SARS-CoV2 challenge. (C) – Viral load measured by TCID 50 titration in lungs of immunized mice on day 3 post SARS-CoV2 challenge. (D) – Survival after SARS-CoV2 challenge. (E) – Weight loss after SARS-CoV2 challenge.

Article Snippet: Final IVT products were purified with the mRNA Clean & Concentrator Kit (ZYMO #R1018).

Techniques: Immunopeptidomics, Titration

Journal: bioRxiv

Article Title: Circular mRNA against CleanCap linear mRNA vectors: comprehensive comparison, expression, active and passive immunization

doi: 10.1101/2025.10.13.682008

Figure Lengend Snippet: I n vivo kinetics curve of B11-Fc antibody expression by the linear and the circular mRNA vectors..

Article Snippet: Final IVT products were purified with the mRNA Clean & Concentrator Kit (ZYMO #R1018).

Techniques: Expressing

Journal: bioRxiv

Article Title: Circular mRNA against CleanCap linear mRNA vectors: comprehensive comparison, expression, active and passive immunization

doi: 10.1101/2025.10.13.682008

Figure Lengend Snippet: Survival test after 5LD 50 BonT/A challenge of treated mice. A – mRNA treatment 4 hours before toxin challenge, B – mRNA treatment 16 hours before toxin challenge, C – mRNA treatment 24 hours before toxin challenge, D – mRNA treatment 48 hours before toxin challenge.

Article Snippet: Final IVT products were purified with the mRNA Clean & Concentrator Kit (ZYMO #R1018).

Techniques: