Journal: Scientific Reports

Article Title: Intravenous high mobility group box 1 fragment improves cardiac function, fibrosis, and coronary flow in porcine ischemic cardiomyopathy model

doi: 10.1038/s41598-026-38592-7

Figure Lengend Snippet: RT-PCR analysis. Comparison of mRNA expression in the border and remote zones between the two groups using RT-PCR. (7A) SDF1, (7B) HGF, (7C) VEGFα, (7D) FGF2, (7E) TGFβ3, (7F) IL-10, (7G) PDGFRα, (7H) PDGFRβ. *p < 0.05, **p < 0.01.

Article Snippet: The following genes were analyzed using TaqMan Gene Expression Assay (Applied Biosystems): SDF1 (Ss03391855_m1), HGF (Ss06927387_m1), VEGFA (Ss03393990_m1), FGF2 (Ss03375809_u1), TGFβ3 (Ss03394351_m1), IL-10 (Ss03382372_u1), PDGFRα (Ss04955107_g1), and PDGFRβ (Ss06879561_m1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Comparison, Expressing

Journal: bioRxiv

Article Title: Mechanism of giant magnetic field effect in fluorescence of mScarlet3, a red fluorescent protein

doi: 10.1101/2025.02.26.640351

Figure Lengend Snippet: E. coli expressing red-emitting fluorescent proteins were grown on LB (lysogeny broth) agar plates and then imaged in an epifluorescence microscope. Panels show responses to a 10 mT magnetic field (grey bars). A 595 nm longpass emission filter was used. A) mScarlet-I3, B) mScarlet3, C) mRuby3, D) mSandy2, E) mKate2.

Article Snippet: The additional fluorescent proteins tested were mScarlet-I3 (Addgene #189757), mRuby3 (Addgene #104005), mSandy2 (Addgene #177760), and mKate2 (Addgene #104030).

Techniques: Expressing, Microscopy

Journal: Journal of proteome research

Article Title: Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.

doi: 10.1021/pr5003593

Figure Lengend Snippet: Figure 1. N40K occurrence is independent of the postmortem interval and correlates with the decrease of the U1-70K level in AD. (A) Identification of N40K in the detergent insoluble fraction of AD brain by 1D SDS gel and LC−MS/MS. Ctl: control case. (B) Validation of N40K only in AD (case 8 in Supporting Information Table S1) by Western blotting. (C) No statistically significant difference in postmortem intervals between the N40K negative and positive cases. (D) Western blotting to show reduced U1-70K level in the total homogenate in N40K positive cases. The numbers above the gel indicate the corresponding case numbers in Supporting Information Table S1. (E) Quantitation of the Western blotting intensity in panel D to indicate an inverse correlation of U1-70K in total homogenate with insoluble N40K. Relative intensities of U1-70K and N40K were normalized by setting the maximal values to 100, respectively.

Article Snippet: Fulllength U1-70K and its truncations were cloned from a commercial plasmid (U1-70K-myc-DDK, pCMV6, Origene, RC201713).

Techniques: SDS-Gel, Liquid Chromatography with Mass Spectroscopy, Control, Biomarker Discovery, Western Blot, Quantitation Assay

Journal: Journal of proteome research

Article Title: Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.

doi: 10.1021/pr5003593

Figure Lengend Snippet: Figure 2. Identification of U1-70K and N40K peptides in AD. These peptides are shown in different colors in the protein sequence, together with peptides monitored in LC−SRM.

Article Snippet: Fulllength U1-70K and its truncations were cloned from a commercial plasmid (U1-70K-myc-DDK, pCMV6, Origene, RC201713).

Techniques: Sequencing

Journal: Journal of proteome research

Article Title: Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.

doi: 10.1021/pr5003593

Figure Lengend Snippet: Figure 3. Analysis of the N40K C-terminus by LC−SRM. (A) Work flow of the method. (B) Identification and size confirmation of purified recombinant U1-70K by Western blotting, followed by band excision and in-gel digestion. (C) Identification of a N40K−containing gel band by Western blotting. The 10 kDa ladder was also used for precise alignment of immunoblotting images with stained gel. (D) The intensity ratios of light/heavy (L/H) of U1-70K peptide pairs, leading to the predicted cleavage region.

Article Snippet: Fulllength U1-70K and its truncations were cloned from a commercial plasmid (U1-70K-myc-DDK, pCMV6, Origene, RC201713).

Techniques: Recombinant, Western Blot, Staining

Journal: Journal of proteome research

Article Title: Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.

doi: 10.1021/pr5003593

Figure Lengend Snippet: Figure 4. Analysis of the N40K N-terminus by MS. (A−C) MS/MS spectra for identifying the N-terminal peptide with methionine removal and threonine acetylation in U1-70K, N40K, and purified U1-70K in HEK293 cells, respectively. Matched ions were assigned by arrows indicating specific b or y ions.

Article Snippet: Fulllength U1-70K and its truncations were cloned from a commercial plasmid (U1-70K-myc-DDK, pCMV6, Origene, RC201713).

Techniques: Tandem Mass Spectroscopy

Journal: Journal of proteome research

Article Title: Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.

doi: 10.1021/pr5003593

Figure Lengend Snippet: Figure 5. Determination of N40K C-terminus by Western blotting. (A) Expression of U1-70K N-terminal truncations in HEK293 cells. (B) Analysis of N40K in AD and three recombinant truncations. (C) Diagram of U1-70K domains and predicted N40K cleavage region. (D) N40K truncations covering all possible cleavage products with different C-terminal residues.

Article Snippet: Fulllength U1-70K and its truncations were cloned from a commercial plasmid (U1-70K-myc-DDK, pCMV6, Origene, RC201713).

Techniques: Western Blot, Expressing, Recombinant

Journal: Journal of proteome research

Article Title: Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.

doi: 10.1021/pr5003593

Figure Lengend Snippet: Figure 6. Toxicity analysis of N40K in rat hippocampal primary neurons. (A) Restriction enzyme digestion at the cloning sites by BamHI and EcoRI to show equal levels of the plasmids of U1-70K and six N40K truncations. (B) Western blotting to show similar protein expression in HEK293 cells. (C) Co-transfection of nine plasmids each with RFP by the calcium phosphate method. (D) Quantitative analysis by counting degenerating neurons. Error bars represent standard errors of mean (SEM) of analyzed neurons in duplicated plates of the same batch of neuronal culture. The experiments were repeated three times with different batches of culture, all showing similar results. The asterisks indicate p < 0.05 by the ANOVA and Tukey range test. (E) The MTT assay of neuronal viability upon Lentiviral N40K transfection. Error bars represent SEM. The double asterisks indicate p < 0.01 by Student’s t-test.

Article Snippet: Fulllength U1-70K and its truncations were cloned from a commercial plasmid (U1-70K-myc-DDK, pCMV6, Origene, RC201713).

Techniques: Cloning, Western Blot, Expressing, Cotransfection, MTT Assay, Transfection

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: Nicotinic agonist intensity-based drug-sensing fluorescent reporter (iDrugSnFR) naming, dose–response relations, and residues mutated. Measurements in E. coli lysates (L) or with purified protein (P). ND, not determined. Data for iAChSnFR from Borden et al., 2019 ; data for iNicSnFR3b from Shivange et al., 2019 .

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Purification

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: Dose–response relations on intermediate constructs using E. coli lysate were performed with respective drug partners to identify site-saturation mutagenesis (SSM) winners. ( A–D ) The progenitor biosensor is listed in black. Dashed lines indicate data that did not reach saturation at the concentrations tested; therefore, EC 50 and ∆F max /F 0 could not be determined. Development of ( A ) iDianiSnFR, ( B ) iCytSnFR, ( C ) iCyt_F_SnFR, and ( D ) iCyt_BrEt_SnFR.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Construct, Mutagenesis

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: Intensity-based drug-sensing fluorescent reporter (iDrugSnFR) dose–response relations versus a selected panel of nicotinic agonists. ND, not determined. *, ** EC 50 and ∆F max /F 0 could not be determined from the data ( Figure 3 ). Therefore, the upper limit to the S-slope is estimated from the data at the foot of the dose–response relation.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques:

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: ( A–D ) Relevant EC 50 values for each iDrugSnFR are listed in . Dashed lines indicate dose–response relations that did not approach saturation for the concentration ranges tested; therefore, EC 50 and ∆F max /F 0 could not be determined. ( A ) iDianiSnFR shows preference for dianicline, with some promiscuity for other nicotinic agonists. ( B ) iCytSnFR, ( C ) iCyt_F_SnFR, and ( D ) iCyt_BrEt_SnFR bind their drug partner, but also respond to other nicotinic agonists. Ch, choline; ACh, acetylcholine; Cyt, cytisine; Diani, dianicline; Nic, nicotine; Var, varenicline; 10FC, 10-fluorocytisine; 9Br10EtC, 9-bromo-10-ethylcytisine.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Concentration Assay

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: ( A ) iDianiSnFR shows no fluorescent response to any of the selected endogenous molecules. ( B ) iCytSnFR, ( C ) iCyt_F_SnFR, and ( D ) iCyt_BrEt_SnFR show no response to any of the selected endogenous molecules except 5-HT and DA at concentrations above 100 µM. ATP, adenosine triphosphate; DA, dopamine; GABA, γ-aminobutyric acid; Glu, glutamate; Gly, glycine; Hist, histamine; L-DOPA, levodopa; NE, norepinephrine; 5-HT, serotonin.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques:

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: Top row: exemplar heat traces of iCytSnFR, iCyt_F_SnFR, iCyt_BrEt_SnFR, and iDianiSnFR paired with their drug partners obtained by isothermal calorimetry. The heats for iCytSnFR, iCyt_F_SnFR, and iCyt_BrEt_SnFR were exothermic, while that for iDianiSnFR was endothermic. Middle row: the resulting fits for each iDrugSnFR:drug pair from the integrated heats comprising each series of injections. Bottom row : energy calculations. All iDrugSnFRs show exergonic reactions, but the relative enthalpic and entropic contributions vary among iDrugSnFRs. Data are from three separate runs, mean ± SEM. iDrugSnFR, intensity-based drug-sensing fluorescent reporter.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques:

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: Affinity, occupancy number, and thermodynamic data calculated from isothermal titration calorimetry. Data are the mean ± SEM, three runs.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Isothermal Titration Calorimetry

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: Fluorescence was activated by mixing with the agonists as noted. Stopped-flow data shows a departure from first-order kinetics for this set of intensity-based drug-sensing fluorescent reporter (iDrugSnFRs). iDianiSnFR and iCyt_F_SnFR are fit to a double exponential; iCytSnFR and iCyt_BrEt_SnFR are fit to a single exponential. ( E–H ) Plots of the observed apparent rate constant against [agonist] for the 1 s data obtained in ( A–D ). In ( H ), we have confidence that the k obs shows a maximal value of 40–50 s –1 ; the K d probably lies within twofold of the fitted value.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Fluorescence

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: ( A–D ) Each iDrugSnFR detects its drug partner at the plasma membrane (PM) and endoplasmic reticulum (ER) of HeLa cells at the concentrations sampled. BC, buffer control. SEM of data are indicated by semi-transparent shrouds around traces where trace width is exceeded. ( A ) iDianiSnFR detects dianicline with a return to baseline fluorescence between drug applications. ( B ) iCytSnFR detection at the PM returns to baseline fluorescence between applications, while detection at the ER shows incomplete wash-in and washout. ( C ) iCyt_F_SnFR fluorescence response to the presence of 10-fluorocytisine in the ER also shows an incomplete washout between applications. ( D ) iCyt_BrEt_SnFR detects 9-bromo-10-ethylcytisine with wash-in and washout fluorescence similar to the pattern seen in iDianiSnFR.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Clinical Proteomics, Membrane, Control, Fluorescence

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: Cultured primary mouse hippocampal neurons were transduced with endoplasmic reticulum (ER)- or plasma membrane (PM)-targeted constructs. BC, buffer control. SEM of data are indicated by semi-transparent shrouds around traces where trace width is exceeded. ( A–D ) Each iDrugSnFR detects its drug partner at the PM and ER over the concentrations sampled. ( A ) iDianiSnFR detects dianicline with a return to baseline fluorescence between drug applications. ( B ) iCytSnFR detection at the PM returns to baseline fluorescence between applications, while detection at the ER shows an incomplete washout. ( C ) iCyt_F_SnFR fluorescence response to the presence of 10-fluorocytisine in the ER also shows an incomplete washout between applications. ( D ) iCyt_BrEt_SnFR_ER detects 9-bromo-10-ethylcytisine with a wash-in and decay intermediate between iDianiSnFR and the other two cytisine derivatives.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Cell Culture, Transduction, Clinical Proteomics, Membrane, Construct, Control, Fluorescence

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: Endoplasmic reticulum (ER)-targeted constructs of iDianiSnFR, iCytSnFR, iCyt_F_SnFR, and iCyt_BrEt_SnFR are shown before ( A1–D1 ) and during ( A2–D2 ) exposure to each drug partner. ER-targeted iDrugSnFRs show the reticulated ER and dark ovals corresponding to the nucleus. Plasma membrane (PM)-targeted constructs of the same iDrugSnFRs are shown before ( E1–H1 ) and after ( E2–H2 ) drug introduction. Localization to the PM is robust, with some minimal puncta that may represent inclusion bodies or internal transport.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Construct, Clinical Proteomics, Membrane

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet: Endoplasmic reticulum (ER)-targeted constructs of iDianiSnFR, iCytSnFR, iCyt_F_SnFR, and iCyt_BrEt_SnFR are shown before ( A1–D1 ) and during ( A2–D2 ) exposure to each drug partner. ER-targeted iDrugSnFRs show the reticulated the ER and dark ovals corresponding to the nucleus. Plasma membrane (PM)-targeted constructs of the same iDrugSnFRs are shown before ( E1–H1 ) and after ( E2–H2 ) drug introduction. Localization in the PM is robust, with some minimal puncta that may represent inclusion bodies or internal transport.

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Construct, Clinical Proteomics, Membrane

Journal: eLife

Article Title: Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

doi: 10.7554/eLife.74648

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , iDianiSnFR , This paper , Addgene: 177741 , Obtainable through Addgene.

Techniques: Isolation, Recombinant, Mutagenesis, Plasmid Preparation, Purification, Gel Extraction, Transfection, Software