Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Ubc9 , Dr. Edward Yeh , Addgene plasmid # 20082 , .

Techniques: Recombinant, Plasmid Preparation

Journal: bioRxiv

Article Title: Long-Term Reconfiguration of Dynamic Polarization and Action Potential Initiation in Purkinje Cell Axons

doi: 10.64898/2026.10.12.758508

Figure Lengend Snippet: A , Representative SRRF confocal images of improved ASAP (axonally-targeted ASAP: axASAP) at the soma/dendrite (left) and at a distal axon of a PC (right). B , Fluorescent intensity at soma, dendrite, axon, and presynaptic bouton of original ASAP1 or axASAP. N = 41 (ASAP1) and 39 cells (axASAP1). ***, p < 0.005; *, p < 0.05.

Article Snippet: The coding DNA sequence for ASAP1 was obtained from Addgene (#52519), and some modifications for higher voltage sensitivity (D145D, L146G, S147T, N149R, S150G, and H151D) established in an improved version ASAP3 ( ) were introduced by PCR-mediated mutations (noted here as ASAP3β; ).

Techniques: Confocal

Journal: bioRxiv

Article Title: Long-Term Reconfiguration of Dynamic Polarization and Action Potential Initiation in Purkinje Cell Axons

doi: 10.64898/2026.10.12.758508

Figure Lengend Snippet: A , Representative image of a HEK293T cell expressing axASAP3. B , Voltage-step protocol (holding potential: −70 mV; steps to −100 ∼ +50 mV) and corresponding optical fluorescence responses (ΔF/F) of ASAP1, axASAP1, and axASAP3. C , D , Normalized fluorescence change time course during 100-mV depolarization (C) and repolarization (D), comparing original ASAP1, axASAP1, and axASAP3. Fast (Ƭ fast ) and slow (Ƭ slow ) time constants and the relative amplitude of the fast component (% fast) are indicated for each probe. E , Simultaneous fluorescent voltage imaging (red, 1.5 kHz) and whole-cell current-clamp electrical (black) recordings of a single AP in the PC soma expressing axASAP1 (left) or original ASAP1 (right), illustrating the fast fluorescence recovery of axASAP1 providing better fluorescent tracking of APs.

Article Snippet: The coding DNA sequence for ASAP1 was obtained from Addgene (#52519), and some modifications for higher voltage sensitivity (D145D, L146G, S147T, N149R, S150G, and H151D) established in an improved version ASAP3 ( ) were introduced by PCR-mediated mutations (noted here as ASAP3β; ).

Techniques: Expressing, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Dynamic regulation of cystathionine gamma-lyase expression during normoxia, ischemia, and reperfusion in liver: interplay of transcription factors, microRNAs, and histone modifications

doi: 10.64898/2026.10.15.759434

Figure Lengend Snippet: (a) Schematic showing multiple factors that were considered to shortlist hsa-miR-150-5p and hsa-miR-510-5p as candidate miRNAs that could target human CTH gene. (b and c) Co-transfection of human CTH 3′-UTR constructs with increasing concentrations of either miR-150-5p or miR-510-5p expression plasmids in Huh7 cells resulted in decreased reporter activity and endogenous CTH protein in a concentration-dependent manner. (d and e) Inhibition of endogenous miR-150-5p and miR-510-5p using respective miRNA inhibitors decreased endogenous miRNA levels and increased endogenous CTH protein levels. (f and g) Deletion of miRNA binding sites on human CTH 3′-UTR enhanced its activity. (i and j) RT-PCR followed by Ago2 IP reveals CTH mRNA was highly enriched in miR-150-5p and miR-510-5p overexpressed samples. The luciferase reporter activity was normalized with β-galactosidase activity and the values obtained for each construct were represented as hCTH 3′-UTR activity. All the values in the graphs are represented as mean ± S.D of n = 3 technical replicates. The transfection experiments were repeated at least three times and representative data are shown. Unpaired t -test or one-way ANOVA with Tukey’s multiple correction as a post-hoc test was used for statistical analysis. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns: not significant

Article Snippet: Similarly, to ascertain the effect of over-expression of miRNAs on 3′-UTR activity and endogenous levels of CTH, CTH-3′-UTR reporter construct (400 ng/well) was co-transfected with increasing concentrations of hsa-miR-150-5p (pcDNA3.2/V5 hsa-mir-150 (purchased from Addgene; plasmid # 26310) or hsa-miR-510-5p expression plasmids using PEI reagent.

Techniques: Cotransfection, Construct, Expressing, Activity Assay, Concentration Assay, Inhibition, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Luciferase, Transfection, Analysis, Expression Vector

Journal: bioRxiv

Article Title: Dynamic regulation of cystathionine gamma-lyase expression during normoxia, ischemia, and reperfusion in liver: interplay of transcription factors, microRNAs, and histone modifications

doi: 10.64898/2026.10.15.759434

Figure Lengend Snippet: CTH pro-275 promoter-luciferase reporter construct was co-transfected with increasing concentrations of CREB, Sp1, and NFY-A/B/C expression plasmids in Huh7 cells. (a-c) CTH promoter activity (top panels) and endogenous CTH mRNA levels (middle panels) increased with increasing CREB, Sp1, and NFY-A protein levels (bottom panels). CTH promoter-reporter construct was co-transfected with increasing concentrations of KCREB, Sp1 shRNA, and NFY-DN expression plasmids in Huh7 cells. (d-f) CTH promoter activity (top panels) and endogenous CTH mRNA decreased (bottom panels) with increasing concentrations of KCREB, Sp1 shRNA or NFYDN expression plasmids. The values in the graphs are mean ± S.D of n = 3 technical replicates. The transfection experiments were repeated at least three times and representative data are shown. The statistical significance was determined by one-way ANOVA with Tukey’s multiple correction as a post-hoc test. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns: not significant. NS: non-specific protein band

Article Snippet: Similarly, to ascertain the effect of over-expression of miRNAs on 3′-UTR activity and endogenous levels of CTH, CTH-3′-UTR reporter construct (400 ng/well) was co-transfected with increasing concentrations of hsa-miR-150-5p (pcDNA3.2/V5 hsa-mir-150 (purchased from Addgene; plasmid # 26310) or hsa-miR-510-5p expression plasmids using PEI reagent.

Techniques: Luciferase, Construct, Transfection, Expressing, Activity Assay, shRNA

Journal: bioRxiv

Article Title: Dynamic regulation of cystathionine gamma-lyase expression during normoxia, ischemia, and reperfusion in liver: interplay of transcription factors, microRNAs, and histone modifications

doi: 10.64898/2026.10.15.759434

Figure Lengend Snippet: Huh 7 cells were transfected with core CTH promoter promoter-reporter constructs (WT and mutant) along with β-galactosidase expression plasmid. (a-c) CTH promoter activity upon mutation of the binding sites of CREB, Sp1, and NFY-A in the core promoter region. The mutated bases in CTH promoter are highlighted in red above the plots. Synergistic activity of these TFs was determined by co-transfection of CTH-WT promoter-reporter construct with a combination of two TFs expression plasmids at a time in Huh7 cells. (d) CTH core promoter (WT) activity upon co-overexpression of two TFs. The values in the graphs are mean ± S.D of n = 3 technical replicates. The transfection experiments were repeated at least three times and representative data are shown. The statistical significance was determined by one-way ANOVA with Tukey’s multiple correction as a post-hoc test. ****P<0.0001, ***P<0.001, **P<0.01, ns: not significant

Article Snippet: Similarly, to ascertain the effect of over-expression of miRNAs on 3′-UTR activity and endogenous levels of CTH, CTH-3′-UTR reporter construct (400 ng/well) was co-transfected with increasing concentrations of hsa-miR-150-5p (pcDNA3.2/V5 hsa-mir-150 (purchased from Addgene; plasmid # 26310) or hsa-miR-510-5p expression plasmids using PEI reagent.

Techniques: Transfection, Construct, Mutagenesis, Expressing, Plasmid Preparation, Activity Assay, Binding Assay, Cotransfection, Over Expression

Journal: bioRxiv

Article Title: Dynamic regulation of cystathionine gamma-lyase expression during normoxia, ischemia, and reperfusion in liver: interplay of transcription factors, microRNAs, and histone modifications

doi: 10.64898/2026.10.15.759434

Figure Lengend Snippet: Huh7 cells were subjected to normoxia, ischemia (4 hr), and reperfusion (1 hr) and microRNA expression levels were measured using RT-PCR. (a) Expression levels of miR-150-5p in Huh7 cells in normoxia, ischemia and reperfusion conditions. (b and c) Correlation between miR-150-5p and CTH expression during normoxia/ischemia conditions and ischemia/reperfusion in Huh7 cells. (d) Expression levels of miR-150-5p in Huh7 cells in normoxia, ischemia and reperfusion conditions. (e and f) Correlation between miR-510-5p and CTH expression during normoxia/ischemia conditions and ischemia/reperfusion in Huh7 cells. (g and h) Expression levels of miR-150-5p and miR-510-5p in mice IR liver (n = 3). All the values in the graphs (except panel g and h) are represented as mean ± S.D of n = 3 technical replicates. The in vitro IR experiments were repeated at least three times and representative data are shown. Unpaired t-test or one-way ANOVA with Tukey’s multiple correction as a post-hoc test was used for statistical analysis. ****P<0.0001, ***P<0.001, *P<0.05, ns: not significant

Article Snippet: Similarly, to ascertain the effect of over-expression of miRNAs on 3′-UTR activity and endogenous levels of CTH, CTH-3′-UTR reporter construct (400 ng/well) was co-transfected with increasing concentrations of hsa-miR-150-5p (pcDNA3.2/V5 hsa-mir-150 (purchased from Addgene; plasmid # 26310) or hsa-miR-510-5p expression plasmids using PEI reagent.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Analysis