Journal: PLoS Pathogens

Article Title: The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation

doi: 10.1371/journal.ppat.1009412

Figure Lengend Snippet: Antibodies Used in This Study.

Article Snippet: Nup98 , Atlas Antibodies , Cat# HPA074810.

Techniques:

Journal: bioRxiv

Article Title: Selective degradation of multimeric proteins via chemically induced proximity to TRIM21

doi: 10.1101/2024.01.31.578122

Figure Lengend Snippet: (A) Workflow of CRISPR-suppressor scanning (created with Biorender.com ). (B) Violin plot showing log 2 transformed fold change in the average abundance of each sgRNA comparing A549-TRIM21 D355A treated with ACE (dose escalating from 500 nM to 20 µM) versus vehicle for three weeks. Three independent samples were included in the analyses. Data used for the plots are provided in Table S6. (C) Scatter plot showing log 2 transformed fold change in the average abundance of each NUP98-targeting sgRNA (y axis) in A549-TRIM21 D355A cells treated with ACE versus vehicle for three weeks. The sgRNAs are arrayed by the amino acid position in the NUP98-NUP96 coding sequence on the x axis corresponding to the position of the predicted cut site. Data points represent the mean values of three independent samples. (D) Workflow to isolate ACE-resistant clones from A549-Cas9 cells transduced with the sgRNA targeting NUP98 K752. (E) Concentration-response curves of ACE on the viability of NUP98 mutant clones with IFNγ (10 ng/ml) pretreatment. Data indicate the mean ± s.e.m. of three independent samples. IC50 and 95% CI are shown in Table S1. (F) Immunoblots of indicated proteins in IFNγ-stimulated parental A549 cells and NUP98 mutant clones (R1–R5) that were treated with ACE (10 μM) for 12 h. A representative result was shown from three independent experiments. Uncropped western blot images are provided as a Source Data file. (G) Volcano plot depicting log 2 transformed average fold change of each protein (quantified by label-free proteomics) and −log 10 transformed P value comparing TRIM21-TurboID enriched proteins from the NUP98 mutant clone (R1) treated with ACE (20 µM) versus vehicle for 4 h. Three independent samples were included in the analyses. P values were calculated by unpaired Student’s t-test (two tailed). Data used for the plot are provided in Table S7. (H) Schematic of the PML-GFP-degron assay. (I) The effect of ACE on the levels of indicated PML-GFP-NPC fusion proteins in A549-TRIM21 D355A cells. Data are the mean ± s.e.m. of six independent samples. P values were calculated by unpaired Student’s t-test (two tailed). (J) Confocal microscopy images of PML-GFP-NUP98 domain 2 (green) in A549-TRIM21 D355A cells treat with vehicle or ACE (10 µM). Scale bar: 10 µm. (K) NUP98 APD in the cryo-electron tomography structure of the NPC (PDB: 7R5J).

Article Snippet: The membranes were blocked in 5% nonfat milk PBST solution (0.1% v/v Tween-20) for 30 min and then were sequentially incubated with the primary antibody overnight at 4 °C and the secondary antibody at room temperature for 1 h. The primary antibodies used are as follows: rabbit polyclonal anti-Stat1 (1:5,000, 9172, Cell Signaling Technology, Danvers, MI, USA), rabbit monoclonal anti-Phospho-Stat1 (1:5,000, 7649, Cell Signaling Technology, Danvers, MI, USA), mouse monoclonal anti-Flag-HRP (1:10000, A8592, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-β-actin-HRP (1:10,000, HX18271, Huaxingbio, Beijing, China), rabbit monoclonal anti-TRIM21 (1:5000, AB207728, Abcam, Cambridge, UK), rabbit polyclonal anti-GLE1 (1:5000, A13207, ABclonal, Woburn, MA, USA), anti-SMPD4 (1:5000, A15473, ABclonal, Woburn, MA, USA), anti-NUP35 (1:5000, A12762, ABclonal, Woburn, MA, USA), and anti-NUP98 (1:5000, A0530, ABclonal, Woburn, MA, USA).

Techniques: CRISPR, Transformation Assay, Sequencing, Clone Assay, Transduction, Concentration Assay, Mutagenesis, Western Blot, Two Tailed Test, Confocal Microscopy, Tomography

Journal: bioRxiv

Article Title: Selective degradation of multimeric proteins via chemically induced proximity to TRIM21

doi: 10.1101/2024.01.31.578122

Figure Lengend Snippet: (A) The cDNA sequences of NUP98 mutant clones. (B) The deleted segment in NUP98 mutant clones highlighted in the structure of NUP98 APD (PDB: 2Q5Y). (C) Volcano plot depicting log 2 transformed average fold change of each protein (quantified by label-free proteomics) and −log 10 transformed P value comparing the NUP98 mutant clone (R1) treated with ACE (10 µM) versus vehicle for 8 h. Three independent samples were included in the analyses. P values were calculated by unpaired Student’s t-test (two tailed). Data used for the plots are provided in Table S8. (D) The effect of ACE on the levels of indicated PML-GFP-NUP98 fusion proteins in A549-TRIM21 D355A cells. Data are the mean ± s.e.m. of six independent samples.

Article Snippet: The membranes were blocked in 5% nonfat milk PBST solution (0.1% v/v Tween-20) for 30 min and then were sequentially incubated with the primary antibody overnight at 4 °C and the secondary antibody at room temperature for 1 h. The primary antibodies used are as follows: rabbit polyclonal anti-Stat1 (1:5,000, 9172, Cell Signaling Technology, Danvers, MI, USA), rabbit monoclonal anti-Phospho-Stat1 (1:5,000, 7649, Cell Signaling Technology, Danvers, MI, USA), mouse monoclonal anti-Flag-HRP (1:10000, A8592, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-β-actin-HRP (1:10,000, HX18271, Huaxingbio, Beijing, China), rabbit monoclonal anti-TRIM21 (1:5000, AB207728, Abcam, Cambridge, UK), rabbit polyclonal anti-GLE1 (1:5000, A13207, ABclonal, Woburn, MA, USA), anti-SMPD4 (1:5000, A15473, ABclonal, Woburn, MA, USA), anti-NUP35 (1:5000, A12762, ABclonal, Woburn, MA, USA), and anti-NUP98 (1:5000, A0530, ABclonal, Woburn, MA, USA).

Techniques: Mutagenesis, Clone Assay, Transformation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Selective degradation of multimeric proteins via chemically induced proximity to TRIM21

doi: 10.1101/2024.01.31.578122

Figure Lengend Snippet: (A) Chemical structure of TrimTAC1. (B) Representative images of A549-TRIM21 D355A or A549-CRBN cells expressing indicated reporter proteins treated with or without TrimTAC1 (4 µM) or dBET1 (100 nM) for 4 h. Scale bar: 10 µM. (C) Concentration-response curves of TrimTAC1 or dBET1 on the normalized intensities of indicated reporter proteins in A549-TRIM21 D355A or A549-CRBN cells. Data indicate the mean ± s.e.m. of three independent samples. IC50 and 95% CI are shown in Table S1. (D) Intensities of NUP98 FG -mEGFP-BRD4 BD2 in A549-TRIM21 D355A cells treated with TrimTAC1 (2 µM) for indicated time. Each point represents the mean of three independent samples. One-way ANOVA followed by Dunnett’s multiple comparison test was used to determine statistical significance. (E) Normalized intensities of NUP98 FG -mEGFP-BRD4 BD2 in A549-TRIM21 D355A cells with indicated treatment of TrimTAC1 (4 µM), BTZ (400 nM), and TAK243 (1.5 µM). Data indicate the mean ± s.e.m. of six independent samples. One way ANOVA followed by Tukey’s multiple comparison test was used to determine statistical significance. (F) Chemical structure of TrimTAC2. (G) Representative images of indicated FKBP12-mEGFP reporter proteins treated with vehicle, TrimTAC1 (10 μM), or TrimTAC2 (10 μM) for 4 h. Scale bar: 10 µm. (H) Schematic illustration of TrimTAC selectivity towards multimeric proteins in biomolecular condensates.

Article Snippet: The membranes were blocked in 5% nonfat milk PBST solution (0.1% v/v Tween-20) for 30 min and then were sequentially incubated with the primary antibody overnight at 4 °C and the secondary antibody at room temperature for 1 h. The primary antibodies used are as follows: rabbit polyclonal anti-Stat1 (1:5,000, 9172, Cell Signaling Technology, Danvers, MI, USA), rabbit monoclonal anti-Phospho-Stat1 (1:5,000, 7649, Cell Signaling Technology, Danvers, MI, USA), mouse monoclonal anti-Flag-HRP (1:10000, A8592, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-β-actin-HRP (1:10,000, HX18271, Huaxingbio, Beijing, China), rabbit monoclonal anti-TRIM21 (1:5000, AB207728, Abcam, Cambridge, UK), rabbit polyclonal anti-GLE1 (1:5000, A13207, ABclonal, Woburn, MA, USA), anti-SMPD4 (1:5000, A15473, ABclonal, Woburn, MA, USA), anti-NUP35 (1:5000, A12762, ABclonal, Woburn, MA, USA), and anti-NUP98 (1:5000, A0530, ABclonal, Woburn, MA, USA).

Techniques: Expressing, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: Selective degradation of multimeric proteins via chemically induced proximity to TRIM21

doi: 10.1101/2024.01.31.578122

Figure Lengend Snippet: (A) Representative images of A549 cells expressing NUP98 FG -mEGFP-BRD4 BD2 pretreated with vehicle, bortezomib (400 nM), or TAK-243 (1.5 µM) for 2 h followed by TrimTAC1 (4 µM) treatment for 4 h. Scale bar: (B) Representative images of A549 cells expressing indicated reporter proteins treated with or without TrimTAC2 (10 µM) for 4 h. Scale bar: 10 μm. (C) Representative images of A549 cells expressing NUP98 FG -mEGFP-FKBP12 pretreated with vehicle, bortezomib (200 nM), or TAK-243 (1 µM) for 2 h followed by TrimTAC2 (10 µM) treatment for 4 h. Scale bar: (D) Concentration-response curves of TrimTAC2 on the intensities of indicated reporter proteins in A549-TRIM21 D355A cells. Data indicate the mean ± s.e.m. of three independent samples. IC50 and 95% CI are shown in Table S1. (E) Intensities of NUP98 FG -mEGFP-FKBP12 in A549-TRIM21 D355A cells with indicated treatment of TrimTAC2 (10 µM), bortezomib (200 nM), and TAK243 (1 µM). Data indicate the mean ± s.e.m. of six independent samples. One way ANOVA followed by Tukey’s multiple comparison test was used to determine statistical significance.

Article Snippet: The membranes were blocked in 5% nonfat milk PBST solution (0.1% v/v Tween-20) for 30 min and then were sequentially incubated with the primary antibody overnight at 4 °C and the secondary antibody at room temperature for 1 h. The primary antibodies used are as follows: rabbit polyclonal anti-Stat1 (1:5,000, 9172, Cell Signaling Technology, Danvers, MI, USA), rabbit monoclonal anti-Phospho-Stat1 (1:5,000, 7649, Cell Signaling Technology, Danvers, MI, USA), mouse monoclonal anti-Flag-HRP (1:10000, A8592, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-β-actin-HRP (1:10,000, HX18271, Huaxingbio, Beijing, China), rabbit monoclonal anti-TRIM21 (1:5000, AB207728, Abcam, Cambridge, UK), rabbit polyclonal anti-GLE1 (1:5000, A13207, ABclonal, Woburn, MA, USA), anti-SMPD4 (1:5000, A15473, ABclonal, Woburn, MA, USA), anti-NUP35 (1:5000, A12762, ABclonal, Woburn, MA, USA), and anti-NUP98 (1:5000, A0530, ABclonal, Woburn, MA, USA).

Techniques: Expressing, Concentration Assay, Comparison

Journal: Life Science Alliance

Article Title: Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect

doi: 10.26508/lsa.202201603

Figure Lengend Snippet: (A) Rif1–mKO2 cells (KYP1866 and KYP1867), in which the endogenous Rif1 was tagged with mKO2, harboring pREP41– Flag 3 vector (upper) or pREP41– Rif1– Flag 3 (lower) were grown in the absence of thiamine for 20 h, and were extracted by Triton X-100 and DNase I and remaining endogenous Rif1–mKO2 signals (mazenta) were observed. The nuclear envelope was stained with Nup98 antibody (green). (B, C) The numbers (B) and the intensities (C) of nuclear foci were quantified in Rif1–mKO2 cells harboring pREP41– Flag 3 (Vector) or pREP41– Rif1– Flag 3 (Rif1OE) grown as in (A). Source data are available for this figure.

Article Snippet: Nup98, a marker of the nuclear membrane, was detected with rat anti-Nup98 monoclonal antibody (1:500; Bioacademia) for 12 h at 4°C after blocking in PBS containing 3% BSA and 0.1% Tween 20.

Techniques: Plasmid Preparation, Staining

Journal: Life Science Alliance

Article Title: Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect

doi: 10.26508/lsa.202201603

Figure Lengend Snippet: (A, B, C) Rif1–mKO2 cells harboring vector ((A) and left panel of (C)) (KYP1866) or Rif1-overexpressing plasmid ((B) and right panel of (C)) (KYP1867) were pretreated with Triton X-100 and DNase I and stained with anti-Nup98 antibody (green; nuclear membrane) and Hoechst 33342 (blue; nuclei). The Hoechst signal is very low because of prior treatment with DNase I. In (A, B), mKO2 signals are in red, whereas they are in black in (C). Strong telomere signals of Rif1–mKO2 are detected in vector control, whereas multiple nuclear foci are detected upon overexpression of Rif1. This reflects its hetero-oligomerization with the overexpressed Rif1 protein and binding to chromosome arms. Source data are available for this figure.

Article Snippet: Nup98, a marker of the nuclear membrane, was detected with rat anti-Nup98 monoclonal antibody (1:500; Bioacademia) for 12 h at 4°C after blocking in PBS containing 3% BSA and 0.1% Tween 20.

Techniques: Plasmid Preparation, Staining, Membrane, Control, Over Expression, Binding Assay

Journal: Life Science Alliance

Article Title: Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect

doi: 10.26508/lsa.202201603

Figure Lengend Snippet: Reagents & Resources.

Article Snippet: Nup98, a marker of the nuclear membrane, was detected with rat anti-Nup98 monoclonal antibody (1:500; Bioacademia) for 12 h at 4°C after blocking in PBS containing 3% BSA and 0.1% Tween 20.

Techniques: Recombinant, Protease Inhibitor, Membrane, Purification, Multiplex Assay, Software

Journal: bioRxiv

Article Title: Residual blinking-driven channel alignment for multicolor single-molecule localization microscopy

doi: 10.64898/2025.12.23.696332

Figure Lengend Snippet: (a) STORM image of Nup98 in Channel 1, immunolabeled with Alexa647-conjugated secondary antibodies. (b) STORM image of Nup62 in Channel 2, immunolabeled with CF583R-conjugated secondary antibodies. (c) STORM image of Channel 3, generated by simultaneous excitation at 647 nm and 561 nm, utilizing residual blinking. (d) Conventional alignment of Channels 1 and 2. The enlarged yellow box highlights an individual NPC (i), and the localization count profiles along the white dashed line (ii). The enlarged view in the white box (iii) and colocalization assessment by cross-correlation analysis (iv), are also shown. (e) Alignment of Channels 1 and 3 via direct cross-correlation. The enlarged views within the yellow box along with the localization count profiles (ii), are shown. The enlarged views within the white box (iii) along with colocalization assessment result (iv), are also shown. (f) Alignment of Channels 2 and 3 performed similarly as in (e). (g) ReBling alignment of Channels 1 and 2. The enlarged yellow box highlights an individual NPC (i), and localization count profiles (ii), are shown. The enlarged view in the white box (iii) and colocalization assessment result (iv), are also shown.

Article Snippet: Mouse monoclonal antibodies of Nup98 were from Cell Signaling Technology (USA).

Techniques: Immunolabeling, Generated