Journal: bioRxiv

Article Title: A surrogate marker of protection confirms the efficacy of an AddaS03-adjuvanted West Nile virus subunit vaccine

doi: 10.64898/2026.04.20.719748

Figure Lengend Snippet: ( A ) Identification of purified sEnv by CBB staining and immunoblotting using a pan-orthoflavivirus anti-fusion loop monoclonal antibody (clone 4G2). ( B ) Five-week-old BALB/c mice (5-week-old, n=6) were immunized subcutaneously with a vaccine formulation containing various antigen doses (0, 0.1, 0.5, 1, or 5 μg) with or without adjuvant (Alhydrogel or AddaS03) twice at 2 weeks interval. Serum samples were collected at 13 and 35 days after the first immunization. ( C to F ) Binding and neutralizing antibody titers in murine sera after priming ( C and E ) and boosting immunizations ( D and F ) were determined by indirect ELISA using WNV SVP and by a neutralization assay, respectively. Dotted line indicates the LOD. The threshold titer estimated as sufficient to protect against lethal WNV infection in is also indicated as the LOD, since the threshold value was below the LOD. Data are represented as the mean±SD of biological replicates (n=6). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparison test. All statistical differences are determined in comparison with the sEnv alone (gray) or Alhydrogel (blue) group within the same antigen dose (* p <0.01, ** p <0.005, **** p <0.0005). ns, not significant.

Article Snippet: The membranes were blocked with 5% skimmed milk in PBST and incubated with pan-orthoflavivirus anti-fusion loop monoclonal antibody 4G2 (supernatant from D1-4G2-4-15 cells, ATCC) at 4°C overnight.

Techniques: Purification, Staining, Western Blot, Formulation, Adjuvant, Binding Assay, Indirect ELISA, Neutralization, Infection, Comparison

Journal: Nucleic Acids Research

Article Title: RSRC2 is a novel RNA-binding protein that safeguards mitotic fidelity by interacting with the lncRNA C1QTNF1-AS1

doi: 10.1093/nar/gkag229

Figure Lengend Snippet: C1QNTF1–AS1 interacts with RSRC2, an RBP of unknown function. ( A ) Schematic representation of the C1QTNF1–AS1 and C1QTNF1/CTRP1 genomic landscape ( C1QTNF1–AS1 annotated as NR_040 018/NR_040 019 in RefSeq; Gencode gene ENSG00000265096 ; chr17:79019209-79027601, hg38). ( B ) Maximum intensity projections of representative images of C1QTNF1–AS1 exon smRNA FISH in HCT116 showing its nuclear localization. Nuclei were stained with DAPI (magenta) and outlined with a dashed circle. Right panel: quantification of total transcript in the nucleus (N) and cytoplasm (C), solid line represents the mean. N = 3 ( n (HCT116) =398). ( C ) Expression levels of C1QTNF1–AS1 in HCT116 cells following depletion with three LNA gapmers targeting either exon 3 (LNA 1) or first intron of C1QTNF1–AS1 (LNA 2, 3), as measured by qPCR. Primers spanning mature (ex2-3) C1QTNF1–AS1 were used. Results are presented relative to negative control (Ctl A) LNA; N = 3. ( D ) Schematic representation of workflow for the ASO pulldown of C1QTNF1–AS1 in HCT116 cells. Five different ASOs targeting different regions of the C1QTNF1–AS1 locus were used, with luciferase (Luc) ASOs as a negative control. Pulldown efficacy was assessed by qPCR , and proteins were identified using LC-MS analysis (see Materials and methods). ( E ) The volcano plot highlighting proteins enriched in C1QTNF1–AS1 pulldown using ASO 1, 3, and 5 versus Luc. Significant C1QTNF1–AS1 protein interactors are highlighted in red (FDR 5%). ( F ) RIP-qPCR from HCT116 extracts. Left panel: Western blot of RSRC2 in the input and IP samples to show RSRC2 IP efficiency compared to IgG. Right panel: RIP-qPCR showing association of RSRC2 with C1QTNF1–AS1 transcript. GAPDH was used as negative control RNA for RSRC2 RIP. RIP enrichments are presented as % of input RNA (normalized to IgG); N = 3. ( G ) Relative expression of RSRC2 in HCT116 cells following siRNA-mediated depletion of RSRC2, as measured by qPCR. Results are presented relative to control siRNA (Ctl); N = 3. ( H ) Representative western blot showing RSRC2 protein expression in HCT116 cells following siRNA-mediated depletion of RSRC2. β-Tubulin and Ponceau staining were used as loading controls. An asterisk indicates an unspecific RSRC2 protein band. ( I ) Densitometric analysis of RSRC2 levels from panel (H) relative to control siRNA (Ctl); N = 3. ( J ) Interaction intensities between C1QTNF1–AS1 and the indicated proteins show that C1QTNF1–AS1 interacts with RSRC2. In-cell interactions were measured in an incPRINT experiment where MS2-tagged C1QTNF1–AS1 RNA was co-expressed with a set of FLAG-tagged proteins in HEK293T cells harbouring a luciferase detector fused to the MS2 coat protein (MS2CP). Upon the formation of FLAG–protein–RNA–MS2–MS2CP ternary complexes, RNA–protein interactions were measured by luciferase activity . eGFP (enhanced green fluorescent protein) was used as a negative control and PABPC3 (a polyadenylated RBP) was used to control for RNA expression. Xist(C) -MS2 vector was used alongside C1QTNF1AS1 –MS2 as a positive control for RNA–protein interactions. RLU are relative light unit; N = 4. ( K ) Protein expression levels were estimated from horseradish peroxidase ELISA of FLAG-tagged proteins in the same experiment as in panel (J). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm; N = number of cells analysed. An unpaired t -test with Welch’s correction was applied in panels (C), (G), and (I). Unpaired t -test was used in panel (F). * <0.05, **<0.01, and ****<0.0001.

Article Snippet: C1QTNF1–AS1 insert was cloned upstream of the MS2 stem-loop sequence of the 10xMS2 vector using a Gibson assembly cloning kit (E5510S, NEB) at 50°C for 15 min.

Techniques: Staining, Expressing, Negative Control, Luciferase, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Activity Assay, RNA Expression, Plasmid Preparation, Positive Control, Enzyme-linked Immunosorbent Assay