pcdna3 2 v5 dest 3xflag srsf3  (Addgene inc)

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

Journal: bioRxiv

Article Title: Dredd-mediated cleavage of Kenny uncouples the IKK complex from selective autophagy to enable innate immunity

doi: 10.64898/2026.04.24.720600

Figure Lengend Snippet: (A) Adult Canton S flies were fed with 5 % sucrose and 100 µM chloroquine for 16 h. Endogenous Ref(2)P, Kenny and Atg8a levels in lysates from adult intestines were analysed with Western blotting with anti-Ref(2)P, anti-Kenny, anti-Atg8a and anti-Tubulin antibodies, n=3. (B) Adult Canton S flies were fed with 5% sucrose and Ecc15 for 16 h, Ref(2)P and Kenny levels were analysed in lysates from dissected intestines by Western blotting with anti-Ref(2)P, anti-Kenny, and anti-Actin antibodies. The relative protein levels of endogenous Ref(2)P and Kenny levels were quantified, n>4. (C) Adult Canton S and Atg8a-GFP flies were fed with 5% sucrose and Ecc15 for 16 h before whole flies were lysed and GFP-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-Kenny, anti-Atg8a, and anti-Actin antibodies. The ratio of immunoprecipitated Ref(2)P and Kenny to total Ref(2)P and Kenny was quantified, n=3. (D) Adult wildtype Canton S and GFP-Kenny expressing flies were fed with 5% sucrose and Ecc15 for 16 h before lysis. GFP-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-GFP, and anti-Actin antibodies. The ratio of immunoprecipitated Ref(2)P to total Ref(2)P was quantified, n=3.

Article Snippet: The following antibodies were used: anti-GFP (ab6556, Abcam), anti-HA (clone 3F10, #11867423001, Roche), anti-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad), anti-Ref(2)P (ab178440, Abcam), anti-Ref(2)P, and anti-Actin (C-11, sc-1615, Santa Cruz).

Techniques: Western Blot, Immunoprecipitation, Expressing, Lysis

Journal: bioRxiv

Article Title: Dredd-mediated cleavage of Kenny uncouples the IKK complex from selective autophagy to enable innate immunity

doi: 10.64898/2026.04.24.720600

Figure Lengend Snippet: (A) Dissected intestines from adult flies expressing mCherry-Atg8a (magenta) crossed with flies expressing HA-tagged wildtype or D21E mutant Kenny were stained for HA (yellow) and Ref(2)P (cyan). Co-localisation of Atg8a, Kenny and Ref(2)P is indicated as white puncta in the merged image. Midguts were imaged by confocal microscopy using at least 3 intestines per repeat, n=3. Scale bar 100 µm. (B) Adult control UbiGal4 flies and flies expressing HA-tagged wildtype or D21E mutant Kenny by control of UbiGal4 were fed with 5% sucrose and Ecc15 for 16 h before lysis. HA-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-HA and anti-Actin antibodies, n=5. The relative interaction between Ref(2)P and Kenny were quantified. (C) Adult control DaGal4 flies and flies expressing V5-tagged wildtype or Δ21 mutant Kenny by control of DaGal4 were lysed and Kenny expression levels were analysed with Western Blotting with anti-Kenny (upper panel), anti-V5 (middle panel), and anti-Actin antibodies, n>3.

Article Snippet: The following antibodies were used: anti-GFP (ab6556, Abcam), anti-HA (clone 3F10, #11867423001, Roche), anti-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad), anti-Ref(2)P (ab178440, Abcam), anti-Ref(2)P, and anti-Actin (C-11, sc-1615, Santa Cruz).

Techniques: Expressing, Mutagenesis, Staining, Confocal Microscopy, Control, Lysis, Western Blot