Journal: iScience

Article Title: tANCHOR-cell-based assay for monitoring of SARS-CoV-2 neutralizing antibodies rapidly adaptive to various receptor-binding domains

doi: 10.1016/j.isci.2024.109123

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Article Snippet: Rabbit anti-V5 , Novus Biologicals , Cat# NB600-381.

Techniques: Virus, Recombinant, Saline, Staining, Isolation, Protease Inhibitor, Software, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell Host & Microbe

Article Title: Determinants of Spike infectivity, processing, and neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2

doi: 10.1016/j.chom.2022.07.006

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Article Snippet: Thereafter, samples were washed with PBS and incubated for 2 h at 4°C with primary antibody (anti-V5(Mouse) (1:1,000, Cell Signalling, #80076S)) diluted in PBS.

Techniques: Recombinant, Mutagenesis, Binding Assay, Plasmid Preparation, Software, Microscopy, Modification

Journal: bioRxiv

Article Title: Influenza A virus NS1 sequesters RNA:DNA hybrids to evade RNase H1-dependent innate immunity

doi: 10.64898/2026.05.04.722684

Figure Lengend Snippet: A . Schematic representation of the modified cytoDRIP-seq approach. A549 cells were infected with IAV at an MOI of 1 PFU/cell for 24h. Immunoprecipitations were performed with anti-V5 (control) or S9.6 antibodies. ΔDRIP-seq represents RNase H1-sensitive RNA:DNA hybrid signal calculated as non-RNase H1-treated samples minus RNase H1-treated samples. Three independent experiments were performed and analyzed. B-C. Total number of reads mapped to the (B) human genome and the (C) IAV genome. Data represent the composition of non-RNase H1-treated samples to illustrate the initial distribution of captured sequences. The dashed horizontal line indicates the minimum threshold for high-confidence signal detection (109 reads); data points below this line represent background noise or low-complexity libraries. Bars represent the mean of n=3 independent biological replicates, with individual dots indicating the values for each replicate. Error bars represent standard deviations. D-E . Bar plots showing the average distribution of normalized signal across human genomic features including coding sequence (CDS), exons, introns, untranslated regions (UTR), and intergenic regions. Data are represented as the mean fold change in signal density relative to the mock condition for (D) V5- and (E) S9.6-immunoprecipitated samples. Dots represent n=3 biological replicates derived from background-subtracted (ΔDRIP-seq) signal. Error bars represent standard deviations. F . Genomic distribution of viral-origin RNA:DNA hybrid sequences mapping to each of the eight IAV genome segments. Coverage plots show the ΔRPM signals (RPM difference between non-RNase H1-treated and RNase H1-treated samples; negative values set to zero) obtained from IAV-infected cells following immunoprecipitation with either S9.6 or V5 (control) antibodies. Overlaid traces in varying shades represent each of the n=3 independent replicates. Genome position is indicated on the x-axis.

Article Snippet: For immunoprecipitation, 10 μL Protein G Dynabeads (10004D; Thermo Fisher) were incubated with 10 μg of mouse monoclonal anti-RNA:DNA hybrid [S9.6] antibody (ENH001; Kerafast) or mouse anti-V5 antibody (MCA1360; Bio-Rad) in 1x binding buffer (20□mM Tris-HCl pH□8.0, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate) for 4-6□h at 4°C.

Techniques: Modification, Infection, Control, Sequencing, Immunoprecipitation, Derivative Assay

Journal: bioRxiv

Article Title: Influenza A virus NS1 sequesters RNA:DNA hybrids to evade RNase H1-dependent innate immunity

doi: 10.64898/2026.05.04.722684

Figure Lengend Snippet: A-B . Immunofluorescence microscopy analysis (A) and associated colocalization analysis (B) of A549 cells infected, or mock, with IAV at an MOI of 1 PFU/cell for 24 h, followed by methanol fixation, proteinase K treatment, and staining for RNA:DNA hybrids (S9.6; green), NS1 (magenta), and DNA (DAPI; blue). Colocalization between NS1 and RNA:DNA hybrids was assessed by determining the Pearson’s correlation coefficient ( r ) between green and magenta signals. Boxplots indicate the minimum to maximum range (whiskers) and mean (horizontal line) r values from n=3 independent experiments. Each dot represents the r value from a single field of view. Non-merged images are presented in Supplementary Figure 3. C-D . Detection of dsRNA (9D5; green), NS1 (magenta), and DNA (DAPI; blue) (C), as well as corresponding colocalization analysis (D) in A549 cells treated as detailed in A-B. Non-merged images are presented in Supplementary Figure 3. E-F . Detection of RNA:DNA hybrids (S9.6; green), PB2 (magenta), and DNA (DAPI; blue) (E), as well as corresponding colocalization analysis (F) in A549 cells treated as detailed in A-B. Non-merged images are presented in Supplementary Figure 3. G-H . Detection of dsRNA (9D5; green), PB2 (magenta), and DNA (DAPI; blue) (G), as well as corresponding colocalization analysis (H) in A549 cells treated as detailed in A-B. Non-merged images are presented in Supplementary Figure 3. I . Immunoblot analysis following immunoprecipitation with specific antibodies. A549 cells were infected with IAV, or mock, at an MOI of 1 PFU/cell for 24 h followed by immunoprecipitation (IP) of resulting cell lysates with anti-V5, anti-dsRNA (9D5), or anti-RNA:DNA hybrid (S9.6) antibodies. Input cell lysate and IP fractions were analyzed by immunoblotting for NS1 and total IgG. J . Immunofluorescence microscopy analysis of A549 cells infected with wt IAV, IAV ΔNS1, or mock, at an MOI of 1 PFU/cell for 24 h, prior to processing as described in A. Upper row shows staining for RNA:DNA hybrids (S9.6; green), dsRNA (9D5; magenta), and DNA (DAPI; blue). Bottom row shows staining for IAV NP (red) and DNA (DAPI; blue). K . Quantification of cytoplasmic RNA:DNA hybrid signals (S9.6; green) from experiments described in J. Boxplots indicate the range and mean log 2 FC of the area fraction occupied by signal relative to mock-infected controls from n=3 independent experiments. Each dot represents the average log 2 FC from multiple fields imaged per well. Dotted line indicates no change relative to mock. For all panels, data are representative of at least n=3 independent experiments. For microscopy images, scale bars represent 10 µm. For panels B, D, F, and H, statistical significance was determined by Mann-Whitney U test (* P□ ≤□0.05; ** P□ ≤□0.01).

Article Snippet: For immunoprecipitation, 10 μL Protein G Dynabeads (10004D; Thermo Fisher) were incubated with 10 μg of mouse monoclonal anti-RNA:DNA hybrid [S9.6] antibody (ENH001; Kerafast) or mouse anti-V5 antibody (MCA1360; Bio-Rad) in 1x binding buffer (20□mM Tris-HCl pH□8.0, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate) for 4-6□h at 4°C.

Techniques: Immunofluorescence, Microscopy, Infection, Staining, Western Blot, Immunoprecipitation, MANN-WHITNEY

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

Article Title: Synapse-specific catecholaminergic modulation of neuronal glutamate release.

doi: 10.1073/pnas.2420496121

Figure Lengend Snippet: Fig. 3. Type I MNs OA receptors include OAMB, which is enriched in the Ib MN and needed to boost locomotion. (A) RNAseq shows expression of five OA receptors in type I MNs. The dashed line shows the maximum normalized counts of any gene observed in attp2 controls. Error bars are ± S.E.M (B) Example image of an NMJ showing a maximum projection confocal image of OAMB receptor staining (green) in a Ib and Is MN pair. Animals had the OAMB receptor CRISPR tagged with 10 copies of the V5 epitope. Staining was against the V5 epitope. (Scale bar, 5 μm.) (C) Bar graph shows the mean ± 95% CI of the mean OAMB staining levels (A.U. arbitrary units) per Ib and Is MNs. Each Ib and Is were paired on the same muscle (10 NMJs, 1 per animal). (**** P <0.0001 by the paired t-test). (D) Mean velocity (mm/s) of control larvae (attp2) (black, n = 15) and animals with OAMB knocked down in type I MNs (oambRNAi; blue, n = 14) crawling in the dark (black bar) followed by under 505 nm light (blue bar). Recording starts at 5 min., following an adjustment period. (E) Mean total displacement for each animal in (D). Attp2 (black, n = 15 animals), oambRNAi (blue, n =14 animals). Red points are mean ± S.E.M. (*** P <0.001; ** P <0.01; n.s., not significant by Student’s paired t-test).

Article Snippet: For OAMB imaging, rat anti- V5 epitope (Novus NBP2- 81037) was used at 1:500.

Techniques: Expressing, Staining, CRISPR, Control

Journal: Nature Communications

Article Title: Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response

doi: 10.1038/s41467-021-27451-w

Figure Lengend Snippet: a Coimmunoprecipitation of myc-tagged NUP210 with SUN1 and SUN2. b Colocalization of SUN2 and myc-tagged NUP210. Scale bar = 5 μm. c Distribution of SUN1 and SUN2 in Nup210 KO (KO-N13) 4T1 cells. Scale bar = 10 μm. d Quantification of SUN2 and SUN1 intensity in Nup210 KO 4T1 cells. Mann–Whitney U test, error bar represents median with an interquartile range. ‘ n ’ equals the number of cells analyzed. e Distribution of Lamin B1 and Lamin A/C in Nup210 KO 4T1 cells. MIP maximum intensity projection. Scale bar = 5 μm. f Quantification of Lamin B1 and Lamin A/C intensity in Nup210 KO 4T1 cells. Mann–Whitney U test, error bar represents median with an interquartile range. ‘ n ’ equals the number of cells analyzed. g 3D reconstruction of Lamin B1, Lamin A/C, and DAPI (heterochromatin foci) distribution in Nup210 KO 4T1 cells. Scale bar = 2 μm. h Subcellular fractionation of LINC complex proteins and mechanosensitive MRTF-A and YAP in Nup210 KO 4T1 cells. i Co-IP of H3.1/3.2 with LINC complex proteins SUN2, BRD4 short isoform (BRD4-SF), and RRP1B in 4T1 cells. j Co-IP of V5-tagged BRD4-SF with NUP210 in 4T1 cells. k Co-IP of H3.1/3.2 and SUN2 in human MCF7 and MDA-MB-231 cell line. l Co-IP of H3.1/3.2 with NUP210 and SUN2 in JQ1-treated 4T1 cells. m Distribution of H3.1/3.2 and H3K27me3 in 4T1 cells treated with bromodomain inhibitor JQ1. Scale bar = 5 μm. n Quantification of H3.1/3.2 and H3K27me3 intensity in JQ1-treated 4T1 cells. Mann–Whitney U test, error bar represents median with an interquartile range. ‘ n ’ equals the number of cells analyzed. o Live-cell tracking of nuclear size in DMSO control ( n = 122) and JQ1-treated 4T1 cells ( n = 127). p qRT-PCR of NUP210-regulated genes in JQ1-treated 4T1 cells. Two-tailed t test, mean ± s.e.m. n = 3 biological replicates. q qRT-PCR of NUP210-regulated genes in BRD4-SF knockdown 4T1 cells. Two-tailed t test, mean ± s.e.m. n = 3.

Article Snippet: Primary antibodies and their dilutions were as follows: NUP210 (1:500; Bethyl Laboratories), β-actin (1:10,000; Abcam), Lamin B1 (1:5000; Abcam), Lamin A/C (1:1000; Abcam), Myc-Tag (1:1000; Cell Signaling Technology), rabbit V5-Tag (1:1000; Cell Signaling Technology), mouse V5-Tag (1:1000; Cell Signaling Technology), RRP1B (1:1000; Millipore-Sigma), SUN1 (1:500; Abcam), SUN2 (1:1000; Abcam), FAK (1:5000; Abcam), p-FAK(Y397) (1:5000; Abcam), p-FAK (Y397) (1:5000; Thermo Fisher Scientific), CCL2 (1:1000; Proteintech), H3.1/3.2 (1:1000; Active Motif), H3K27me3 (1:5000; Cell Signaling Technology), H3K9me3 (1:5000; Abcam), SUV39H1 (1:1000, Cell Signaling Technology), EZH2 (1:1000; Cell Signaling Technology), and SUZ12 (1:1000; Cell Signaling Technology).

Techniques: MANN-WHITNEY, Fractionation, Co-Immunoprecipitation Assay, Cell Tracking Assay, Control, Quantitative RT-PCR, Two Tailed Test, Knockdown