Journal: bioRxiv

Article Title: RIG-I-like receptor-dependent type I Interferon regulates antigen dose and activation in yellow fever vaccine 17D-infected antigen presenting cells

doi: 10.1101/2025.10.08.680190

Figure Lengend Snippet: (A) Wildtype 1205Lu cells were infected with YF17D (MOI 1). At 24, 48,72 hrs p. i. cells were fixed, stained with anti-GAPDH (green), anti-dsRNA antibodies (red) and DAPI nuclear staining (blue) and analyzed by confocal microscopy. Representative images are shown. Scale bar, 50 µm, n=2. (B) RNA isolated from YF17D-infected 1205Lu cells at 48 hrs post infection was digested with the indicated enzymes and transfected into 1205Lu cells. CXCL10 concentrations were measured in the supernatants after 24 hrs by ELISA. One-Way ANOVA test with Dunnett’s correction was performed between control and treated samples (n=4, except RiboShredder condition where n=3; ***p<0.001). (C) RNA was isolated from YF17D-infected 1205Lu cells at 24 and 72 hrs post infection and treated with 5PP or RNAse III and then transfected into 1205Lu cells. CXCL10 levels were measured in the supernatants after 24 hrs. Results are shown as percentages of the untreated controls (w/o enzyme) from each experiment, n=2. (D) Schematic depiction of the lPL approach. (E) 1205Lu cells expressing N-mSAv-RIG-I were infected with 10 MOI YF17D for 48 hrs (one experiment). After infection, RIG-I/RNA complexes were isolated using streptavidin pull-down. The purified RNA was used to generate cDNA libraries, which were analyzed by strand specific NGS analysis. The read sequences were aligned to the YF genome reference in sense (blue) or antisense orientation (red). The y-axis depicts genome coverage; the x-axis represents positions on the reference genome.

Article Snippet: The isolated RNA was incubated with streptavidin beads for 4 hrs at 4°C, and the specific RIG-I/RNA complexes recovered by streptavidin pull-down were checked for quality using an RNA 6000 Pico chip (Bioanalyzer 2100, Agilent) and analyzed by NGS.

Techniques: Infection, Staining, Confocal Microscopy, Isolation, Transfection, Enzyme-linked Immunosorbent Assay, Control, Expressing, Purification

Journal: bioRxiv

Article Title: [ 123 I]Italia: A PARP-Directed Auger Electron-Emitting Agent for Targeted Radionuclide Therapy of Cancer

doi: 10.64898/2026.03.13.711622

Figure Lengend Snippet: Pulldown assay demonstrating binding of [ 123 I]Italia_A to PARP and damaged DNA in the presence or absence of talazoparib. A ) Pull down from PARP. B ) Pulldown from DNA.

Article Snippet: Streptavidin-based pull-down assays using Thermo ScientificTM PierceTM Streptavidin Agarose (according to the manufacturer’s instructions) was used to isolate btDNA and His-PARP complexes were isolated by anti-His immunoprecipitation using Protein G Agarose Resin 4 Rapid Run.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

doi: 10.64898/2026.02.23.707416

Figure Lengend Snippet: ( A ) Top: putative STAT sites in the EBI3 and IL12A promoters. Bottom: ChIP-qPCR analysis of STAT3 recruitment to the indicated regions of EBI3 and IL12A promoters in BMDCs at 0.5 h after LDPm infection ( n = 6 replicates). Results are presented as fold enrichment relative to uninfected BMDCs. ( B ) Left: Details of EBI3 and IL12A promoter-specific oligonucleotides containing wild-type or mutated STAT sites (mutated bases in italics) used for the DNA pull-down assay. Right: DNA pull-down analysis using streptavidin (SA)-conjugated Dynabeads, followed by immunoblotting to assess the binding of STAT3 [present in the nuclear lysates of LDPm-infected (0.5 h) BMDCs] to the biotin (Btn)-labeled oligonucleotides shown in the left panel (representative of n = 3 experiments). ( C ) Immunoblot analysis confirming STAT3 silencing by siRNA; β-actin serves as a loading control (representative of n = 3 experiments). Ctrl siRNA, control siRNA. ( D ) IL-35 expression in uninfected and LDPm-infected BMDCs (48 h infection) transfected with the indicated siRNAs, analyzed by flow cytometry [representative data (left) and compiled data (right) from n = 3 experiments]. ( E ) Effect of TIM-3 blockade using an anti-TIM-3 antibody on IL-35 production by BMDCs infected with LDPm for 48 h, analyzed by flow cytometry [representative (left) and compiled (right) data from n = 3 experiments]. Uninfected BMDCs without any antibody treatment (no Ab) serve as controls. Each symbol represents data from one replicate (A) or one experiment (right panels of D and E). Horizontal bars (right panels of D and E) denote means; error bars (A) indicate SD. *** P < 0.001; ns, not significant.

Article Snippet: The Dynabeads M-280 Streptavidin for DNA pull-down assays was from Thermo Fisher Scientific.

Techniques: ChIP-qPCR, Infection, Pull Down Assay, Western Blot, Binding Assay, Labeling, Control, Expressing, Transfection, Flow Cytometry

Journal: Science Advances

Article Title: YAP/TAZ-VGLL3 governs adipocyte fate via epigenetic reprogramming of PPARγ and its target enhancers

doi: 10.1126/sciadv.aea7235

Figure Lengend Snippet: ( A ) The 3D structure of human TAZ (UniProt accession Q9GZV5 ) predicted by AlphaFold is shown on the left. The domain organization of human TAZ is shown on the right: TEAD binding domain (TBD), WW domain, coiled-coil (CC) domain, transactivation domain (TAD), and PDZ binding motif. Serine-to-alanine (SA) mutation sites for constitutively active mutants (red circles) and a TEAD-binding–deficient mutant (blue circle). ( B and C ) Streptavidin pull-down assay for human embryonic kidney (HEK) 293T cells showing the interaction of green fluorescent protein (GFP)–tagged PPARγ2 (B) or endogenous TEADs and LATS1 (C) with SFB (S protein–FLAG–streptavidin binding peptide)–tagged TAZ mutants. ( D to F ) C3H10T1/2 cells stably expressing doxycycline-inducible 2xHA-tagged TAZ mutants were subjected to immunoblot analysis (D), RT-qPCR analysis ( n = 3) (E), and lipid staining with oil red O (F). Scale bars, 200 μm (F). ( G ) MA plot showing differential H3K27ac ChIP-seq signals between control and TAZ2SA-expressing C3H10T1/2 cells (left) and heatmaps showing H3K27ac ChIP-seq signals for cells expressing the indicated TAZ2SA mutants (right). Significantly changed regions (|fold change| > 2 and adj. P value of <0.05) in TAZ2SA-expressing versus control cells are colored red (fold change > 2) or blue (fold change < −2). ( H ) Genome browser views of the indicated loci showing the coverage of the H3K27ac ChIP-seq signal in differentiated C3H10T1/2 cells expressing the TAZ mutants. ( I ) Heatmap of the H3K27ac ChIP-seq signals from C3H10T1/2 cells expressing indicated TAZ mutants, aligned with PPARγ ChIP-seq peaks (D6). Data in bar graph (E) are means ± SEM and analyzed by one-way ANOVA with Tukey’s post hoc test. ** P < 0.01 and *** P < 0.001.

Article Snippet: For streptavidin-mediated pull-down assay of S protein–FLAG–streptavidin binding peptide (SFB)–tagged proteins, cleared cell lysates (1 mg of protein in 1 ml) were incubated for 2 hours at 4°C with 20 μl of Pierce High Capacity Streptavidin Agarose (20359, Thermo Fisher Scientific), and the beads were then washed three times with lysis buffer and boiled with Laemmli sample buffer for immunoblot analysis.

Techniques: Binding Assay, Mutagenesis, Pull Down Assay, Stable Transfection, Expressing, Western Blot, Quantitative RT-PCR, Staining, ChIP-sequencing, Control

Journal: Science Advances

Article Title: YAP/TAZ-VGLL3 governs adipocyte fate via epigenetic reprogramming of PPARγ and its target enhancers

doi: 10.1126/sciadv.aea7235

Figure Lengend Snippet: ( A ) Scatter plot showing correlations between RNA fold change and ATAC gene activity change in adipocyte-related cells (LAKO versus control; sn sequencing). Color indicates the maximum percentage of cells expressing each gene. ( B ) Vgll3 expression in iWAT snRNA-seq. Dediff., Dedifferentiated adipocytes. ( C ) Genomic browser view of Vgll3 locus with the indicated sequencing data. ( D ) Mouse adipose tissue RNA-seq data ( GSE138911 ) showing Vgll3 expression [fragments per million mapped reads (FPM)] in high-fat diet (HFD) or normal chow (NC)–fed adipocyte-specific YAP/TAZ KO (YTKO) mice ( Yap1 fl/fl ; Wwtr1 fl/fl ; Adipoq-Cre ). ( E ) Human visceral adipose tissue RNA data from the GTEx consortium showing the correlation between WWTR1 and VGLL3 expression (right). ( F ) C3H10T1/2 cells expressing doxycycline (Dox)–inducible HA-TAZ2SA were treated with 2 μM VT-104 for 36 hours and subjected to RT-qPCR. ( G ) C3H10T1/2 cells (D6) expressing Dox-inducible Myc-Vgll3 were analyzed by RT-qPCR ( n = 3) and immunoblot. ( H ) Oil red O staining of Dox-inducible Vgll3 or Vgll3ΔTDU-expressing cells. Scale bars, 200 μm. ( I ) Vgll3 KO C3H10T1/2 cells expressing Dox-inducible HA-TAZ2SA and its parental cells (Con) were analyzed by RT-qPCR and immunoblot. ( J ) Genomic view of Pparg (left) and Fabp4 (right) regions with the indicated ChIP-seq data from TAZ mutant–expressing cells. ( K ) H3K27ac peak heatmap aligned with PPARγ ChIP-seq peaks (D6). ( L ) Streptavidin pull-down assay of HEK293T cells cotransfected with HA-tagged histone deacetylase 3 (HDAC3) and SFB-tagged VGLL3. ( M ) C3H10T1/2 cells with inducible Vgll3 expression (TRE-Vgll3) and Hdac3 [short hairpin Hdac3 (shHdac3)] or Ncor1 (shNcor1) knockdown or parental control (−) were treated with adipogenic cocktails and Dox or vehicle control (48 hours) and subjected to RT-qPCR. ( N ) A proposed model for the role of TAZ in adipocyte differentiation and dedifferentiation. Data in bar graphs (D, E, and G) are means ± SEM and analyzed by the unpaired t test. ** P < 0.01 and *** P < 0.001.

Article Snippet: For streptavidin-mediated pull-down assay of S protein–FLAG–streptavidin binding peptide (SFB)–tagged proteins, cleared cell lysates (1 mg of protein in 1 ml) were incubated for 2 hours at 4°C with 20 μl of Pierce High Capacity Streptavidin Agarose (20359, Thermo Fisher Scientific), and the beads were then washed three times with lysis buffer and boiled with Laemmli sample buffer for immunoblot analysis.

Techniques: Activity Assay, Control, Sequencing, Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Staining, ChIP-sequencing, Mutagenesis, Pull Down Assay, Histone Deacetylase Assay, Knockdown

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dynamic extracellular interactions with AMPA receptors

doi: 10.1073/pnas.2517436122

Figure Lengend Snippet: IgLONs interact with the extracellular region of AMPARs. ( A ) Differential biotinylation of IgLON proteins by APEX2-AMPARs identified by BioSITe-MS. Four Iglons identified are listed. Number of unique peptides identified and total number of BxxP-modified peptides (No. shown in parentheses) are shown for each gene, as well as the site of biotinylation (tyrosine, Y). *LTP/Ctrl, P ≤ 0.05, # LTP/AP5 P ≤ 0.05. ( B ) Western blot analysis of streptavidin-enriched lysates after APEX labeling by APEX2-AMPARs in neurons. Left , Immunoblotting with antibodies against IgLON proteins OBCAM, NTM, and Kilon/NEGR1 detects bands in streptavidin-enriched fractions only in samples where APEX2-AMPARs were expressed (lane 2). No IgLONs were detected in fractions isolated from control labeled neurons where APEX2 was not expressed (lane 1). Immunoblots for GluA2 and GluA1 are shown as positive controls. Right , immunoblotting with anti-biotin antibodies shows robust APEX labeling only when APEX2-AMPARs are expressed (right two lanes). ( C ) AMPARs interact with OBCAM in HEK cells. HA-tagged OBCAM was expressed in HEK cells along with individual, Myc-tagged AMPAR subunits (GluA1-4). AMPAR complexes were immunoprecipitated (co-IP) from whole-cell lysates using anti-myc antibodies. Immunoblotting with anti-HA antibodies show OBCAM is present in AMPAR precipitates, but not in controls (wild-type or cells empty pCAG vector). Immunoblotting of whole-cell lysates ( Lower panels) shows relative expression of each AMPAR subunit and OBCAM prior to co-IP. ( D ) OBCAM directly interacts with GluA2-containing AMPARs. Co-IP experiments in HEK cells show HA-OBCAM is present in GluA2 precipitates (lane 4). To demonstrate specificity of this interaction we used a CRISPR KO guide targeting OBCAM cDNA. Expression of OBCAM is successfully depleted by expression of CRISPR knockout (KO) guides targeting the cDNA (OBCAM KO g#2) (lane 2) and thereby depletes OBCAM from GluA2 precipitates (lane 5). ( E ) IgLONs interact with endogenous AMPAR complexes in neurons. Neurons expressing HA-tagged OBCAM or NTM were fractionated and endogenous AMPARs were purified from crude membrane fractions using anti-GluA1 antibodies. Immunoblotting with anti-HA antibodies shows HA-tagged IgLONs precipitate with GluA1 AMPARs, but not in control neurons. HA-IgLONs were found in AMPAR complexes isolated using anti-GluA2 antibodies specific for the N- and C terminus ( SI Appendix , Fig. S2 C and D ). ( F ) Schematic of IgLON-AMPAR complexes. IgLONs are GPI-anchored proteins with all 3 protein domains expressed on the cell surface. Picture shows IgLONs interacting with AMPARs in cis (on the same side of the membrane). It is possible that they interact with AMPARs in cis or trans (across the synapse/from another membrane).

Article Snippet: Cells were solubilized for 2 h at 4 °C, and lysates were cleared by centrifugation at 17,000× g for 20 min. For streptavidin pull-downs, 250 μg labeled neuronal lysates were mixed with 75 μL streptavidin magnetic beads (Thermo Fisher #88817) that had been washed 3×.

Techniques: Modification, Western Blot, Labeling, Isolation, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, Expressing, CRISPR, Knock-Out, Purification, Membrane