Journal: Cell systems

Article Title: Molecular and Functional Networks Linked to Sarcopenia Prevention by Caloric Restriction in Rhesus Monkeys

doi: 10.1016/j.cels.2019.12.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were rinsed thoroughly then incubated in biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG (Vector Labs) for 1 h. Sections were rinsed, then incubated in peroxidase labeled avidin biotin complex (ABC) solution (Vector Labs) for 1 h and color developed with ImPACT NovaRED reagent (Vector Labs) for ~5–8 min and rinsed.

Techniques: Plasmid Preparation, Recombinant, Avidin-Biotin Assay, Electron Microscopy, Sample Prep, Microarray, Labeling, Software

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Cox4i2 deficiency reduces inflammation in mice after 8 months of cigarette smoke exposure. a-d) FACS analysis of bronchoalveolar lavage (BAL) from mice exposed to cigarette smoke (CS) or room air (RA) for 8 months ( n =4 per group): Numbers of neutrophils (a), CD3 + T cells (b), CD4 + T cells (c) and CD8 + T cells (d). e-h) Immunofluorescence analysis of CD45 + (e, f) and CD3 + (g, h) cell accumulation in the lung parenchyma of mice exposed to CS or RA for 8 months ( n =4 per group). e, g: Quantification. f, h: Representative images. i-l) Microarray analysis of lung homogenates from mice exposed to CS or RA for 8 months ( n =6 per group). Most differentially regulated pathways between Cox4i2 −/− and WT mice (i). Differentially expressed genes involved in inflammatory pathways: NF-κB signalling (j), cytokine–cytokine receptor interaction (k) and T-cell receptor signalling (l). m) Multiplex analysis of BAL fluid (BALF) from mice exposed to CS or RA for 8 months ( n =5 per group). Statistical analysis was performed using two-way ANOVA. Data from panels a-e, g and m were log-transformed prior to statistical analysis. The data are presented as the mean ± SEM.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Immunofluorescence, Microarray, Multiplex Assay, Transformation Assay

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Cox4i2 is predominantly expressed in pericytes, smooth muscle cells and fibroblasts a) Schematic illustration of sample acquisition and data generation from CD45 + and CD45 - cells for single-cell RNAseq analyses. b) UMAP representation of CD45 - cells ( n =2–3 per group). c) UMAP highlighting Cox4i2 expressing cells in the pericyte/ smooth muscle cells (SMC) cluster of CD45 - cells. d) DotPlot visualization of marker genes for pericytes, SMCs and fibroblasts (FBs). e) Protein expression of COX4I2 in primary lung FBs, pulmonary artery SMCs (PASMCs), alveolar type II cells (ATIIs), endothelial cells (ECs), pericyte pellet, T cells, neutrophils and macrophages. PASMCs isolated from Cox4i2 −/− mice served as a negative control. f) Track plot of pericyte markers and fibroblast markers g) UMAP representation of the expression of Cox4i2 , Acta2 , or both in pericytes. Red: cells expressing Acta2 ; green: cells expressing Cox4i2 ; yellow: cells expressing both Acta2 and Cox4i2 . h) DotPlot visualization of marker genes expressed in pericytes. i) Labelling of Cox4i2 -expressing cells was performed using Cox4i2 -CreERT2-tdTomato mice. A Kozak-CreERT2-P2A cassette was inserted just before the Cox4i2 start codon, and these mice were crossed with tdTomato reporter mice containing a loxP-STOP-loxP cassette. After tamoxifen (i.p.; TAM) injection, Cre activation removed the stop cassette, enabling tdTomato expression driven by the Cox4i2 promoter. j, k) Representative images. j: tdTomato-positive cells in alveolar structures costained with CD31 as an endothelial marker and ACTA2 as an SMC marker. k: tdTomato-positive cells in pulmonary vessels. Colour code: Red – tdTomato, green – ACTA2, bright cyan – CD31, blue – Hoechst.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Single Cell, RNA sequencing, Expressing, Marker, Isolation, Negative Control, Injection, Activation Assay

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Cell clusters identified by scRNA-seq of CD45 - and CD45 + lung cells after 3 months of CS exposure a) UMAP representation of CD45 + cells (n=2–3 per group). b, c) Numbers of different clusters of CD45 - (b) and CD45 + (c) cells.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Cell marker genes of CD45 - and CD45 + lung clusters a) DotPlot visualization of the gene expression of different cell markers in the CD45 - clusters. b) DotPlot visualization of the gene expression of different cell markers in the CD45 + clusters.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Marker, Gene Expression

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: Expression of Cox4i2 in different cell clusters identified by scRNA-seq of CD45 - and CD45 + lung cells a-c) Annotation strategy of neutrophils at different developmental stages (a) by analysing pseudotime trajectory (b) and violin plots of gene expression of different neutrophil markers (c): Cd101 + neutrophils ( Cd101, Csf3r, and Il1b ), Ngp + neutrophils ( Ngp, Ltf, and Camp ), and Treml4+ neutrophils ( Treml4, Cx3cr1, and Clec4a1 ). d-f) Schematic illustration of the developmental stage of monocytes (d); pseudotime trajectory of monocytes (e) and violin plots of monocyte and macrophage markers (f). Monocyte/macrophage clusters expressing the classical marker Cd68 were classified on the basis of distinct marker expression: alveolar macrophages ( Plet1, Lpl, and Hebp1 ), interstitial macrophages ( C1qa, C1qb, and C1qc ), Cd300e + macrophages ( Ace, Cd300e, and Dusp16 ), monocytes ( Fn1, F13a1, and Ccr2 ), and transitional monocyte/macrophage clusters ( Nr4a1, Apoc2, and Clec4a1 ), which coexpress markers of nonclassical monocytes and maladaptive macrophages. g) Expression of Cox4i2 mRNA across different cell clusters within CD45 - and CD45 + cell populations.

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Expressing, Gene Expression, Marker

Journal: bioRxiv

Article Title: Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

doi: 10.64898/2026.02.09.703513

Figure Lengend Snippet: a) WT mice were exposed to room air (RA) or cigarette smoke (CS) for 8 months, followed by treatment with MitoQ (50 mg/kg/day) or the inactive carrier decylTPP+ for 3 months via gavage. b) Lung functional analyses ( n =7-8 per group) of static compliance. c-d) Stereological analysis of the lung parenchyma ( n =6 per group) showing the number of alveoli (c) and representative pictures (d). e) Haemodynamic measurements ( n =8 per group): right ventricular systolic pressure (RVSP). f, g) Morphological analysis of pulmonary vessels ( n =5 per group): Data are provided for fully, partially, or nonmuscularized vessels as a percentage of the total vessel count. f: Quantification. g. Representative images. h, i) Immunofluorescence analysis ( n =4 per group) of CD45 + cell accumulation in the lung parenchyma. h: Quantification. i: Representative images. j, k) 3-Nitrotyrosine staining of lung tissue ( n =4 per group). j: Quantification of nitrotyrosine staining. k: Representative images. l, m) Percentage of EdU-positive cells in mouse PCLS treated with 5% CSE in the presence of either MitoQ or DecylTPPLJ ( n =3 per group). l: Quantification of EdU-positive cells. m: Representative images. Statistical analysis was performed using one-way and two-way ANOVA. Data from panels h were log-transformed prior to statistical analysis. The data are presented as the mean ± SEM. n, o) Summary of findings showing the critical role of COX4I2 in CS-induced pulmonary inflammation and emphysema development and the therapeutic potential of MitoQ (n) and COXI2-dependent regulation of neutrophil migration via pericytes (o).

Article Snippet: Cells were MACS sorted by labelling with biotin-conjugated antibodies against CD45 (#130-110-657, Miltenyi Biotec), CD31 (#130-111-353, Miltenyi Biotec), CD326 (#130-117-751, Miltenyi Biotec), and CD140a (#130-101-502, Miltenyi Biotec) for 15 minutes at 4°C.

Techniques: Functional Assay, Immunofluorescence, Staining, Transformation Assay, Migration

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Ketoconazole and Posaconazole Selectively Target HK2-expressing Glioblastoma Cells

doi: 10.1158/1078-0432.CCR-18-1854

Figure Lengend Snippet: HK2 loss results in suppression of several oncogenic signaling pathways. A, Western blot analysis was performed 72 hours post-siRNA transfection to confirm HK2 knockdown in multiple GBM cell lines and GSC. Western blot analysis on HK1 was performed as a control to confirm HK2 siRNA specificity. B, Hierarchical clustering of 1,087 genes that were up- or downregulated with HK2 siRNA treatment greater than 1.5-fold based on microarray results. Microarray analysis was performed on 2 cell lines (U87, GSC30) with 2 biological replicates for each. R1 and R2 stand for biological replicates 1 and 2, respectively. C, RT-PCR analysis was performed to confirm HK2 mRNA decrease in HK2 siRNA–treated cells. This analysis was performed on 3 biological replicates and 3 technical replicates for each treatment condition indicated and data are presented as the average of decrease in 4 cell lines. D, Top 200 differentially expressed mRNAs with HK2 siRNA treatment in the U87 and GSC30 cell lines were subjected to pathway analysis using Cytoscape. Blue circles denote downregulated pathways, whereas red circles denote upregulated processes including hypoxia, metabolism, and blood vessel formation.

Article Snippet: HK2 rabbit mAb (2876, Cell Signaling Technology) was used at a 1:300 dilution.

Techniques: Protein-Protein interactions, Western Blot, Transfection, Knockdown, Control, Microarray, Reverse Transcription Polymerase Chain Reaction

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Ketoconazole and Posaconazole Selectively Target HK2-expressing Glioblastoma Cells

doi: 10.1158/1078-0432.CCR-18-1854

Figure Lengend Snippet: Gene signature associated with HK2 knockdown identified several small-molecule inhibitors that can target cells expressing high levels of HK2. A, Schematic of C-Map that was utilized for analysis of genes that were up- and downregulated with HK2 knockdown. B, List of top 15 drugs with a negative enrichment score from C-Map analysis that were predicted to inhibit gene signatures associated with HK2 knockdown. Cells were treated with the identified inhibitors at 1 μmol/L (C) and 10 μmol/L (D) and their viability was assessed using an ATP-cell titer GLO viability assay. The average viability of 3 independent experiments is shown and is depicted as a heatmap. E, Results from yeast drug screen on HXK2 (HK2 homolog) and HXK2 knockout (KO) identified ketoconazole as a potential inhibitor of HXK2, as it did not cause a fitness defect in HXK2 knockout yeast.

Article Snippet: HK2 rabbit mAb (2876, Cell Signaling Technology) was used at a 1:300 dilution.

Techniques: Knockdown, Expressing, Viability Assay, Knock-Out

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Ketoconazole and Posaconazole Selectively Target HK2-expressing Glioblastoma Cells

doi: 10.1158/1078-0432.CCR-18-1854

Figure Lengend Snippet: Ketoconazole and posaconazole inhibit GBM growth in xenograft mouse models. Kaplan–Meier survival analysis of vehicle-treated, ketoconazole-treated (25 mg/kg), and posaconazole-treated (25 mg/kg) mouse GBM models intracranially injected with GSC 8–18 (A) and GSC7–2 (B) cells. C and D, Histopathologic analysis of GBMs extracted from mice treated with azoles by hematoxylin and eosin (H&E), TUNEL, Ki-67, and HK2 staining. E, Quantification of Ki-67–positive cells in azole-treated and vehicle-treated GBM xenografts as an indication of proliferating cells indicated a decrease in proportion of proliferating cells in treated tumors. F, Quantification of TUNEL-positive cells in treated and untreated GBM xenograft models as a measure for proportion of apoptotic cells suggested that treatment with both ketoconazole and posaconazole increased the percentage of apoptotic cells in tumors. G, Quantification of HK2 to determine the effect of azoles of HK2 protein level in tumor sections. All experiments were performed with a minimum of 3 biological replicates and 3 technical replicates. Asterisks on graphs denote a significant difference (*, P < 0.05, **, P < 0.01).

Article Snippet: HK2 rabbit mAb (2876, Cell Signaling Technology) was used at a 1:300 dilution.

Techniques: Injection, TUNEL Assay, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Ketoconazole and Posaconazole Selectively Target HK2-expressing Glioblastoma Cells

doi: 10.1158/1078-0432.CCR-18-1854

Figure Lengend Snippet: Ketoconazole and posaconazole reduce metabolism in GBM cells. A, 13C glucose–labeled flux analysis in GSC 8–18 cells treated with vehicle (DMSO; V), ketoconazole (K), and posaconazole (P) for 24 hours and quantification of glycolytic intermediates. B and C, Quantification of relative hexokinase (HK) activity and extracellular lactate production in GSC 8–18 cells treated with ketoconazole (Keto) or posaconazole (Posa), compared with vehicle-treated cells (V) at 24 hours. Alamar blue cell viability analysis on drug-treated and vehicle-treated GSC 8–18 (D) and GSC 7–2 (E) cells transfected with HK2 siRNA or scrambled siRNA at 72 hours. All experiments were performed with a minimum of 3 biological replicates and 3 technical replicates. Asterisks on graphs denote a significant difference (*, P < 0.05, **, P < 0.01). KD, knockdown.

Article Snippet: HK2 rabbit mAb (2876, Cell Signaling Technology) was used at a 1:300 dilution.

Techniques: Labeling, Activity Assay, Transfection, Knockdown

Journal: Cell systems

Article Title: Molecular and Functional Networks Linked to Sarcopenia Prevention by Caloric Restriction in Rhesus Monkeys

doi: 10.1016/j.cels.2019.12.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were rinsed thoroughly then incubated in biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG (Vector Labs) for 1 h. Sections were rinsed, then incubated in peroxidase labeled avidin biotin complex (ABC) solution (Vector Labs) for 1 h and color developed with ImPACT NovaRED reagent (Vector Labs) for ~5–8 min and rinsed.

Techniques: Plasmid Preparation, Recombinant, Avidin-Biotin Assay, Electron Microscopy, Sample Prep, Microarray, Labeling, Software

Journal: PLoS ONE

Article Title: Local Administration of Soluble CD40:Fc to the Salivary Glands of Non-Obese Diabetic Mice Does Not Ameliorate Autoimmune Inflammation

doi: 10.1371/journal.pone.0051375

Figure Lengend Snippet: HEK293 cells were transduced with AAV2-CD40:Fc or were mock transduced. The fusion protein was detected by anti murine-CD40 (left panel) and anti-human-Fc (right) and showed a band ∼90 kD (size of non-reduced dimer) and a ∼45 kD (reduced monomer) band.

Article Snippet: The blot was blocked with 5% milk powder in TBST buffer and incubated with biotin labeled anti-murine CD40 (R&D systems, MAB4401), followed by streptavidin-HRP (R&D systems) or anti human IgG-HRP (Genetex) and developed with ECL reagent (GE Heatlhcare, Buckinghamshire, UK).

Techniques: Transduction

Journal: PLoS ONE

Article Title: Local Administration of Soluble CD40:Fc to the Salivary Glands of Non-Obese Diabetic Mice Does Not Ameliorate Autoimmune Inflammation

doi: 10.1371/journal.pone.0051375

Figure Lengend Snippet: Microarray analysis was performed on SG of CD40:Fc treated and control mice (n = 4 each, treatment at 10 weeks, end of study at 20 weeks) and gene changes greater than 2 fold were used in the analysis and compared for biological functional pathways or biomarkers by an Ingenuity Pathway analysis (IPA). ( A ) The pathway of NF-κB and TGF-β was affected in CD40:Fc treated mice. Green colored shapes indicates upregulated genes, red colored shapes downregulated; gray colored shapes genes in the pathway that were not differentially expressed. Color intensity reflects the degree of differential expression. Triangles represent phosphatases; horizontal diamond, peptidases; vertical diamond, enzymes; horizontal oval, transcription factors; vertical oval, transmembrane receptors; trapezoid, transporters; circle, other type of protein. Dotted lines indicate an indirect interaction; solid line, direct interaction, solid arrow head indicates “acts on”. ( B ) Metacore analysis of pathways linking differentially expressed genes to immune pathways. The differentially expressed genes are indicated along with the direction of change in expression. ( C ) Quantitative-PCR of selected genes shows agreement with microarray results. The results obtained using the microarray platform were validated by examining the correlation between the expression levels in the microarray and qPCR results obtained for a subset of genes.

Article Snippet: The blot was blocked with 5% milk powder in TBST buffer and incubated with biotin labeled anti-murine CD40 (R&D systems, MAB4401), followed by streptavidin-HRP (R&D systems) or anti human IgG-HRP (Genetex) and developed with ECL reagent (GE Heatlhcare, Buckinghamshire, UK).

Techniques: Microarray, Control, Functional Assay, Quantitative Proteomics, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Local Administration of Soluble CD40:Fc to the Salivary Glands of Non-Obese Diabetic Mice Does Not Ameliorate Autoimmune Inflammation

doi: 10.1371/journal.pone.0051375

Figure Lengend Snippet: Up or down regulated genes in salivary glands of CD40:Fc treated mice versus control mice.

Article Snippet: The blot was blocked with 5% milk powder in TBST buffer and incubated with biotin labeled anti-murine CD40 (R&D systems, MAB4401), followed by streptavidin-HRP (R&D systems) or anti human IgG-HRP (Genetex) and developed with ECL reagent (GE Heatlhcare, Buckinghamshire, UK).

Techniques: Control

Journal: PLoS ONE

Article Title: Local Administration of Soluble CD40:Fc to the Salivary Glands of Non-Obese Diabetic Mice Does Not Ameliorate Autoimmune Inflammation

doi: 10.1371/journal.pone.0051375

Figure Lengend Snippet: FS was determined for each group of CD40:Fc treated mice and compared with control mice (n = 8–10 mice per group). Shown is the average FS per group. Error bar = standard deviation (SD), i.m. = intramuscular.

Article Snippet: The blot was blocked with 5% milk powder in TBST buffer and incubated with biotin labeled anti-murine CD40 (R&D systems, MAB4401), followed by streptavidin-HRP (R&D systems) or anti human IgG-HRP (Genetex) and developed with ECL reagent (GE Heatlhcare, Buckinghamshire, UK).

Techniques: Control, Standard Deviation

Journal: bioRxiv

Article Title: Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response

doi: 10.1101/2024.11.20.623956

Figure Lengend Snippet: A. Domain schematic of the protein PHRF1, highlighting the RING finger (purple oval), PHD finger (brown oval), and SRI region (green rectangle). B. Structural homology model of the PHD region of PHRF1 (shown as a translucent protein surface with electrostatics calculated through APBS in PyMOL) docked to a peptide comprised of the first eight residues of histone H3 (ARTKQTAR), shown as a peptide in brown. P221 of PHRF1 and the first 4 residues of the H3 peptide are also shown as stick representations. This model was generated in Alphafold-Multimer. C. A zoomed in region of the PHRF1 PHD:H3 ( – ) structural homology model highlighting a binding pocket formed by PHRF1 P221 for H3 A1 to enter. D. A sequence schematic for GST-tagged PHRF1 constructs for in vitro experiments. E. Biotinylated histone peptide microarray binding images with GST-PHRF1 PHD (top), GST-PHRF1 RP (middle), and GST-PHRF1 RP(P221L) (bottom). Triplicate red dots indicate a positive histone peptide binding event (each peptide is arrayed in triplicate). Regions outlined by yellow boxes indicate anti-rabbit positive controls. F. A heatmap comparing binding intensities observed in the peptide microarrays for each of the three GST-PHRF1 constructs to select peptides (indicated on the y-axis). Binding strength is represented on a color gradient from red to blue (stronger to weaker) (n=4). For full peptide microarray data, please see Supplementary File 1. G. Representative western blots of biotinylated nucleosomes pulldowns between differentially modified nucleosomes (top) and GST-PHRF1 constructs (right). Anti-GST blots represent PHRF1 signal and anti-H3 blots are shown as loading controls. Key: H3 NΔ2 and NΔ32 are nucleosomes lacking residues 1-2 pr 1-32 on H3, respectively, and H3 tetra-ac = H3 K4acK9acK14acK18ac.

Article Snippet: Separately, 5 μg of anti-PHRF1 antibody (in-house) or normal rabbit IgG (CST; 2729) was incubated with 35 μL of Protein G Dynabeads (ThermoFisher) in lysis buffer containing 1% BSA for 2 hours at 4 °C to immobilize the antibodies.

Techniques: Generated, Binding Assay, Sequencing, Construct, In Vitro, Peptide Microarray, Western Blot, Modification

Journal: bioRxiv

Article Title: Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response

doi: 10.1101/2024.11.20.623956

Figure Lengend Snippet: A. Representative confocal immunofluorescence of control and ΔPHRF1 cells stained with anti-γH2A.X (red), anti-53BP1 (green) antibodies, and DAPI (blue) at indicated time points along the zeocin DDR assay, as described in Supplementary Figure S6A . The scale bar is 10 μm. B, C. Quantitative analysis of γH2A.X ( B ) and 53BP1 ( C ) foci/ nucleus in control (gray) and ΔPHRF1 (mustard green) HCT116 cells. Median values are shown with solid black lines. D, E. qGAM of γH2A.X ( D ) and 53BP1 ( E ) foci/nucleus accumulation over time for control (gray) and ΔPHRF1 (mustard green) cells. Median values are represented as solid, colored circles connected by dashed lines. The qGAM-generated models are represented with smooth lines and shaded in 95% confidence intervals. Statistical significance was determined using a Wald test. **** = p-value < 0.0001. See Supplementary Table S2 for full statistical analysis. F. Representative images of a colony formation assay (CFA) comparing growth in control and ΔPHRF1 HCT116 cells at the indicated concentrations of zeocin. This experiment was conducted in biological triplicate. G. Quantification of growth from the CFA in panel F , comparing control (gray) and ΔPHRF1 (mustard green) HCT116 cells. Statistical significance was determined through a Kruskal-Wallis test; **** = p-value <0.0001, ns = not significant.

Article Snippet: Separately, 5 μg of anti-PHRF1 antibody (in-house) or normal rabbit IgG (CST; 2729) was incubated with 35 μL of Protein G Dynabeads (ThermoFisher) in lysis buffer containing 1% BSA for 2 hours at 4 °C to immobilize the antibodies.

Techniques: Immunofluorescence, Control, Staining, Generated, Colony Assay

Journal: bioRxiv

Article Title: Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response

doi: 10.1101/2024.11.20.623956

Figure Lengend Snippet: A. Representative images of a colony formation assay (CFA) comparing growth in Control+EV (empty vector) and ΔPHRF1 + indicated complemented HCT116 cells at the indicated concentrations of zeocin. This experiment was conducted in biological triplicate. B. Quantification of growth from the CFA in panel A , comparing ΔPHRF1+EV (mustard green) cells to Control+EV (gray), ΔPHRF1+3XFlag-PHRF1 WT (green), and ΔPHRF1+3XFlag-PHRF1 P221L (red) HCT116 cells. Error bars are S.D. Statistical significance was determined through a Kruskal-Wallis test; **** = p-value <0.0001, *** = p-value <0.001, ** = p-value <0.01, * = p-value <0.05, and ns = not significant.

Article Snippet: Separately, 5 μg of anti-PHRF1 antibody (in-house) or normal rabbit IgG (CST; 2729) was incubated with 35 μL of Protein G Dynabeads (ThermoFisher) in lysis buffer containing 1% BSA for 2 hours at 4 °C to immobilize the antibodies.

Techniques: Colony Assay, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response

doi: 10.1101/2024.11.20.623956

Figure Lengend Snippet: A. Schematic of miniTurbo-PHRF1 and the experimental approach for the proximity-based biotinylation assay. B. Volcano plot of the miniTurbo-PHRF1 BioID MS analysis, with splicing-relayed proteins shown in orange, transcription-related proteins shown in mustard green, DDR-related proteins shown in blue, cell cycle-related proteins shown in purple, PHRF1 itself shown in red and labeled, and other significant hits shown in black. The x-axis shows enrichment (log2(fold-change)) of proteins in miniTurbo-PHRF1 samples compared to miniTurbo-GFP and NLS-miniTurbo-GFP negative controls; the y-axis shows significance of enrichment (-log(p-value)). Dashed lines at log2(fold-change) = 1 and p-value = 0.05 signify cutoffs for statistical significance. Experiment done in biological duplicate. C. Pathway analysis of significant hits from the BioID assay using DAVID. The x-axis is fold enrichment, GO terms are ordered in decreasing fold enrichment along the y-axis. The size of each bubble represents gene count and statistical significance is shown as a purple to pink gradient with all terms having a p-value < 0.05.

Article Snippet: Separately, 5 μg of anti-PHRF1 antibody (in-house) or normal rabbit IgG (CST; 2729) was incubated with 35 μL of Protein G Dynabeads (ThermoFisher) in lysis buffer containing 1% BSA for 2 hours at 4 °C to immobilize the antibodies.

Techniques: Cell Surface Biotinylation Assay, Labeling

Journal: bioRxiv

Article Title: Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response

doi: 10.1101/2024.11.20.623956

Figure Lengend Snippet: A. Volcano plot comparing the expression of genes in ΔPHRF1 and control HeLa cells. After applying an FDR cutoff of 0.0001, significant hits that are upregulated in ΔPHRF1 cells are shown in red and significant hits that are downregulated in ΔPHRF1 cells are shown in blue. The x-axis shows fold change in ΔPHRF1 cells over control cells ((log2(fold-change)) and the y-axis shows statistical significance (-log(FDR)). Inset: western blot analysis of PHRF1 protein levels in control and ΔPHRF1 HeLa cells. Experiments were done in biological triplicate. B. Pathway analysis of statistically significant changes in gene expression (FDR <0.0001) using Ingenuity Pathway Analysis (IPA). The x-axis shows activation scores (z-score) of canonical pathways listed on the y-axis, with a positive z-score meaning upregulation and a negative z-score meaning downregulation. Bubble size represents gene ratio and each pathway’s statistical significance is indicated by a color gradient from brown to white. C. Summary pie chart of total alternative splicing identified by rMATS from the significant reads from panel A after filtering out events with an FDR of > 0.05 and Δ4′ values of <0.01. D. Summary table of events represented in panel C . Specifically, the number of differential alternative splicing events as well as the number of corresponding genes are indicated for each of the five annotated splicing event types. E. GO term analysis of differentially alternatively spliced genes from panel D using Enrichr and the GO Biological Process 2023 database. The x-axis shows enrichment of each pathway as gene ratio, the bubble sizes represent gene count, and significance is denoted by a color gradient from blue to white.

Article Snippet: Separately, 5 μg of anti-PHRF1 antibody (in-house) or normal rabbit IgG (CST; 2729) was incubated with 35 μL of Protein G Dynabeads (ThermoFisher) in lysis buffer containing 1% BSA for 2 hours at 4 °C to immobilize the antibodies.

Techniques: Expressing, Control, Western Blot, Activation Assay, Alternative Splicing

Journal: bioRxiv

Article Title: Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response

doi: 10.1101/2024.11.20.623956

Figure Lengend Snippet: A. Experimental schematic of zeocin DDR assay in HeLa cells. Time points indicate when cells were collected for analysis along the experimental trajectory. B. Representative confocal immunofluorescence of control and ΔPHRF1 cells stained with anti-γH2A.X (red) antibody and DAPI (blue) at indicated time points along the zeocin DDR assay. Scale bar is 10 μm. C. Quantitative analysis of γH2A.X intensity/nucleus in control (gray) and ΔPHRF1 (mustard green) cells in the zeocin DDR assay. Median values are shown with solid black lines. D. Quantile general additive modeling (qGAM) of γH2A.X signal accumulation over time from panel C for control (gray) and ΔPHRF1 (mustard green) cells. Median values are represented as solid, colored circles connected by dashed lines. The qGAM generated models are represented with smooth lines and shaded in 95% confidence intervals. Statistical significance was determined using a Wald test. **** = p-value < 0.0001. See Supplementary Table S1 for full statistical analysis.

Article Snippet: Separately, 5 μg of anti-PHRF1 antibody (in-house) or normal rabbit IgG (CST; 2729) was incubated with 35 μL of Protein G Dynabeads (ThermoFisher) in lysis buffer containing 1% BSA for 2 hours at 4 °C to immobilize the antibodies.

Techniques: Immunofluorescence, Control, Staining, Generated

Journal: bioRxiv

Article Title: Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response

doi: 10.1101/2024.11.20.623956

Figure Lengend Snippet: A. Boxplots illustrate distribution of PHRF1 expression levels (log 2 (TPM+1)) in cancer cells from the Cancer Cell Line Encyclopedia (CCLE) collapsed into the indicated tissue types on the y-axis. The central line in each box represents the median. Each cell line’s individual PHRF1 expression is represented as a gray circle. B. Western blot of PHRF1 protein levels in the indicated cancer cell lines and their corresponding cancer type category. C. Volcano plot comparing the expression of genes in ΔPHRF1 and control HCT116 cells. After applying an FDR cutoff of 0.05, significant hits that are upregulated in ΔPHRF1 cells are shown in red and significant hits that are downregulated in ΔPHRF1 cells are shown in blue. The x-axis shows fold change in ΔPHRF1 cells over control cells ((log 2 (fold-change)) and the y-axis shows statistical significance (-log(FDR)). Inset: western blot analysis of PHRF1 protein levels in control and ΔPHRF1 HCT116 cells. Experiments were done in biological triplicate. D. Pathway analysis of statistically significant changes in gene expression (FDR <0.05) using IPA, as in . Bubble size represents gene ratio and each pathway’s statistical significance is indicated by a color gradient from brown to white. E, F. Venn diagram comparison of the number of upregulated ( E ) and downregulated ( F ) pathways identified from HeLa and HCT116 RNAseq experiments.

Article Snippet: Separately, 5 μg of anti-PHRF1 antibody (in-house) or normal rabbit IgG (CST; 2729) was incubated with 35 μL of Protein G Dynabeads (ThermoFisher) in lysis buffer containing 1% BSA for 2 hours at 4 °C to immobilize the antibodies.

Techniques: Expressing, Western Blot, Control, Comparison

Journal: bioRxiv

Article Title: Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response

doi: 10.1101/2024.11.20.623956

Figure Lengend Snippet: A-D. Representative confocal immunofluorescence of Control+EV ( A ), ΔPHRF1+EV ( B ), ΔPHRF1+3XFlag-PHRF1 WT ( C ), and ΔPHRF1+3XFlag-PHRF1 P221L ( D ) cells stained with anti-γH2A.X (red), anti-53BP1 (green) antibodies, and DAPI (blue) at indicated time points along the zeocin DDR assay, as described in Supplementary Figure S6A . The scale bar is 10 μm. E-J. qGAM of γH2A.X ( E-G ) and 53BP1 ( H-J ) foci/nucleus accumulation over time, comparing ΔPHRF1+EV (mustard green) with Control+EV (gray; E, H ), ΔPHRF1+3XFlag-PHRF1 WT (green; F, I ), and ΔPHRF1+3XFlag-PHRF1 P221L (red; G, J ) cells. Median values are represented as solid, colored circles connected by dashed lines. The qGAM-generated models are represented with smooth lines and shaded in 95% confidence intervals. Statistical significance was determined using a Wald test. **** = p-value < 0.0001, ** = p-value <0.01, and ns = not significant. See Supplementary Table S3 for full statistical analysis.

Article Snippet: Separately, 5 μg of anti-PHRF1 antibody (in-house) or normal rabbit IgG (CST; 2729) was incubated with 35 μL of Protein G Dynabeads (ThermoFisher) in lysis buffer containing 1% BSA for 2 hours at 4 °C to immobilize the antibodies.

Techniques: Immunofluorescence, Control, Staining, Generated

Journal: Oncotarget

Article Title: The ribosomal protein S6 in renal cell carcinoma: functional relevance and potential as biomarker

doi:

Figure Lengend Snippet: A further inhibition of Everolimus (1μM) on proliferation A. and protein synthesis B. is abolished in rpS6 depleted cells, but not in 4ebp1 or p27 depleted cells. Note that values are normalized to 100% to allow a direct comparison between different siRNAs. LF = Lipofectamine (mean ±SD; n = 3, *p < 0.05, * *p < 0.01; t -test). Protein synthesis was measured after 35S-methionine labeling.

Article Snippet: The tissue micro array slides were dewaxed and rehydrated using xylene and a series of graded alcohols, followed by heat-induced antigen retrieval using a target retrieval solution (S2031, DakoCytomation, Glostrup, Denmark) in a pressure cooker for 10 min. Staining was performed using an automated staining system (Techmate 500, DakoCytomation) with anti-rpS6 (S6 (5G10) Rabbit mAb #2217, Cell Signaling, Boston, USA) and anti-p-rpS6 (S6-p240 Mouse mAbDAK-S6-240, DakoCytomation, Glostrup, Denmark) for 90 min, and avidin-biotin-complex peroxidase technique using aminoethylcarbazole for visualisation and hematoxylin for counterstaining.

Techniques: Inhibition, Comparison, Labeling

Journal: Oncotarget

Article Title: The ribosomal protein S6 in renal cell carcinoma: functional relevance and potential as biomarker

doi:

Figure Lengend Snippet: Expression of rpS6 (red staining) in normal renal tissue A. and a clear-cell RCC B. , as well as expression of p-rpS6 (brown staining) in normal renal tissue C. and a clear-cell RCC D. was assessed by immunohistochemistry.

Article Snippet: The tissue micro array slides were dewaxed and rehydrated using xylene and a series of graded alcohols, followed by heat-induced antigen retrieval using a target retrieval solution (S2031, DakoCytomation, Glostrup, Denmark) in a pressure cooker for 10 min. Staining was performed using an automated staining system (Techmate 500, DakoCytomation) with anti-rpS6 (S6 (5G10) Rabbit mAb #2217, Cell Signaling, Boston, USA) and anti-p-rpS6 (S6-p240 Mouse mAbDAK-S6-240, DakoCytomation, Glostrup, Denmark) for 90 min, and avidin-biotin-complex peroxidase technique using aminoethylcarbazole for visualisation and hematoxylin for counterstaining.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Oncotarget

Article Title: The ribosomal protein S6 in renal cell carcinoma: functional relevance and potential as biomarker

doi:

Figure Lengend Snippet: Summary of clinical and pathologic features

Article Snippet: The tissue micro array slides were dewaxed and rehydrated using xylene and a series of graded alcohols, followed by heat-induced antigen retrieval using a target retrieval solution (S2031, DakoCytomation, Glostrup, Denmark) in a pressure cooker for 10 min. Staining was performed using an automated staining system (Techmate 500, DakoCytomation) with anti-rpS6 (S6 (5G10) Rabbit mAb #2217, Cell Signaling, Boston, USA) and anti-p-rpS6 (S6-p240 Mouse mAbDAK-S6-240, DakoCytomation, Glostrup, Denmark) for 90 min, and avidin-biotin-complex peroxidase technique using aminoethylcarbazole for visualisation and hematoxylin for counterstaining.

Techniques:

Journal: Oncotarget

Article Title: The ribosomal protein S6 in renal cell carcinoma: functional relevance and potential as biomarker

doi:

Figure Lengend Snippet: Comparison of rpS6 / p-rpS6 expression levels and clinical and pathologic features

Article Snippet: The tissue micro array slides were dewaxed and rehydrated using xylene and a series of graded alcohols, followed by heat-induced antigen retrieval using a target retrieval solution (S2031, DakoCytomation, Glostrup, Denmark) in a pressure cooker for 10 min. Staining was performed using an automated staining system (Techmate 500, DakoCytomation) with anti-rpS6 (S6 (5G10) Rabbit mAb #2217, Cell Signaling, Boston, USA) and anti-p-rpS6 (S6-p240 Mouse mAbDAK-S6-240, DakoCytomation, Glostrup, Denmark) for 90 min, and avidin-biotin-complex peroxidase technique using aminoethylcarbazole for visualisation and hematoxylin for counterstaining.

Techniques: Comparison, Expressing

Journal: Oncotarget

Article Title: The ribosomal protein S6 in renal cell carcinoma: functional relevance and potential as biomarker

doi:

Figure Lengend Snippet: A. Cancer specific survival depending on rpS6 expression levels ( n = 580), patients with low rpS6 expression levels ( n = 387) vs . patients with high rpS6 expression levels ( n = 193). B. Cancer specific survival depending on p-rpS6 expression levels ( n = 598), patients with low p-rpS6 expression levels ( n = 473) vs . patients with high p-rpS6 expression levels ( n = 125). C. Cancer specific survival depending on p-rpS6 expression in patients with low ( n = 339) and high rpS6 ( n = 167) expression. Patients with low rpS6 expressing RCCs are further divided into groups with low ( n = 300) and high ( n = 39) p-rpS6 expression, the same partitioning is done for patients with high rpS6 expression: low p-rpS6 ( n = 103) and high p-rpS6 ( n = 64).

Article Snippet: The tissue micro array slides were dewaxed and rehydrated using xylene and a series of graded alcohols, followed by heat-induced antigen retrieval using a target retrieval solution (S2031, DakoCytomation, Glostrup, Denmark) in a pressure cooker for 10 min. Staining was performed using an automated staining system (Techmate 500, DakoCytomation) with anti-rpS6 (S6 (5G10) Rabbit mAb #2217, Cell Signaling, Boston, USA) and anti-p-rpS6 (S6-p240 Mouse mAbDAK-S6-240, DakoCytomation, Glostrup, Denmark) for 90 min, and avidin-biotin-complex peroxidase technique using aminoethylcarbazole for visualisation and hematoxylin for counterstaining.

Techniques: Expressing

Journal: Oncotarget

Article Title: The ribosomal protein S6 in renal cell carcinoma: functional relevance and potential as biomarker

doi:

Figure Lengend Snippet: Uni- and multivariate analyses of rpS6 and p-rpS6 expression and clinical/pathologic features for the prediction of cancer specific survival in patients with RCCs

Article Snippet: The tissue micro array slides were dewaxed and rehydrated using xylene and a series of graded alcohols, followed by heat-induced antigen retrieval using a target retrieval solution (S2031, DakoCytomation, Glostrup, Denmark) in a pressure cooker for 10 min. Staining was performed using an automated staining system (Techmate 500, DakoCytomation) with anti-rpS6 (S6 (5G10) Rabbit mAb #2217, Cell Signaling, Boston, USA) and anti-p-rpS6 (S6-p240 Mouse mAbDAK-S6-240, DakoCytomation, Glostrup, Denmark) for 90 min, and avidin-biotin-complex peroxidase technique using aminoethylcarbazole for visualisation and hematoxylin for counterstaining.

Techniques: Expressing

Journal: Communications biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts.

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: Fig. 1 Design and strategy toward protein degradation assay on-chip. a pDOC is a MITOMI-based microfluidic platform. The device includes flow (green) and control (Magenta) layers, and several modules: 1) common manifold enabling loading of materials, which in combination with microarray can enable up to 512 different experiments; 2) Parallel loading inputs enabling gel-like loading of up to 32 different experimental conditions; and 3) MITOMI module of 512 cell units, which controls the degradation assay process. b An illustration of two cell units. Each cell unit (marked by black dotted frame) comprises two chambers controlled by three pneumatic integrated valves. The total volume of each unit is about 1 nanoliter. The two chambers are separated by the ‘neck valve’ (I). Different cell units are separated via sandwich valves (II). Samples containing target proteins (IVT products) are loaded into the protein chamber and immobilized via biotinylated antibodies that can be either protein- or tag specific. The target proteins are trapped via MITOMI button valve (III) for quantification while the remaining unbound biomaterials are washed away. Flow direction within the device is indicated by green arrows. c Left, an image of a cell units within the MITOMI array. Target proteins can be immobilized to the protein chamber and quantified in several ways (illustrated on the right): i) An example of a target protein carrying a fluorescent tag (e.g., GFP) for detection (see green glowing tag) and a non-fluorescent tag for immobilization; ii) A target protein tagged with GFP for both immobilization (by anti-GFP antibodies) and detection; iii) The target protein is untagged. Detection is based on fluorescent lysine (Lys) incorporated during in vitro translation. Immobilization is via protein-specific antibodies. iv) The target protein is immobilized via tag- or protein-specific antibodies and detected by immunofluorescence via tag-specific antibodies coupled to fluorophore. Overall, on-chip immobilization of target proteins relies on biotinylated antibodies. Immobilization via non-biotinylated antibodies is possible if surface chemistry includes biotinylated IgG.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated antiGFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Degradation Assay, Control, Microarray, In Vitro

Journal: Communications biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts.

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: Fig. 3 Method versatility. a Degradation of Flag-Securin-Myc, Flag-Securin-GFP, and Green-Lys-labeled Flag-Securin (IVT products) in NDB mitotic extracts was assayed in tube for 1 h. On-chip analysis was performed in multiple ways: 1) Flag-Securin-Myc was immobilized by anti-Myc antibodies and detected by anti-Flag Cy5-conjugated antibodies; 2) Flag-Securin-GFP was immobilized and detected via the GFP tag; and 3) Green-Lys-labeled Flag- Securin was immobilized via anti-Flag antibodies and detected by the Green-Lys signal. Anti-Myc/Flag/GFP antibodies are biotinylated. Plots and raw data depict protein levels at t0 vs. t60 min. Signals are normalized to max values at t0. Mean values and standard error bars are shown; n = 20–40 cell units. *p value < 0.001. b Degradation of Green-Lys-labeled p27 (untagged) and Flag-Securin-GFP was assayed in S-phase extracts and analyzed by pDOC. p27 was immobilized via biotinylated anti-mouse IgG and anti-p27 antibodies. The Green-Lys signal was used for detection. Flag-Securin-GFP was immobilized via biotinylated anti-Flag antibodies. Following incubation with cell extracts, protein levels were quantified by GFP or Green-Lys fluorescence in 15 min intervals. Plots depict mean and standard error values normalized to max signal at t0. n = 20 (p27) and 10 (Securin) cell units.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated antiGFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Labeling, Incubation

Journal: Communications biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts.

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: Fig. 2 On-chip analysis of protein degradation assays. a Time-dependent degradation of 35S-labeled Flag-Securin-GFP, Flag-Geminin-GFP and their non- degradable variants (Δ64 and Δ27, respectively) in NDB mitotic extracts (20 µl) supplemented with E-mix and Ubiquitin. Time-dependent proteolysis was resolved by SDS-PAGE and autoradiography. b Equivalent assays were performed with non-radioactive IVT products. Target proteins were incubated 1 h in reaction solution containing either extracts or PBS (control). Aliquots of each reaction mix (5 µl) were then loaded directly on a chip via separate channels, each containing dozens of cell units. Target proteins were immobilized to protein chambers via biotinylated anti-GFP antibody. The GFP tag was also used for quantification. GFP signals were calculated from 14–19 cell units per target protein per reaction condition (extracts vs. PBS). Box plots depict ratios of GFP signals (extracts/PBS) at t60 min. Mean (x) and median (-) are indicated. *p value < 0.001. Representative raw data depicting detection on chip of the four target proteins are shown. c The degradation of Flag-Securin-GFP variant was assayed in tube as described in B. Here, however, aliquots were snap- frozen every 15 min. Time-lapse samples were then loaded on the chip for analysis. Target proteins were immobilized in protein chambers via anti-GFP antibodies and quantified based on GFP fluorescence. Time-dependent degradations of w.t vs. mutant Securin were quantified based on 35S signal (standard analysis; n = 3] and GFP fluorescence (on-chip analysis). Plots depict mean signals and standard error bars. Mean signals were calculated from 26 cell units and normalized between max (1, t0) and min (0, t60 min) values, allowing proper comparison between two very different methods of detection. Error bars are shown. d Equivalent experiment to (c) performed with Flag-Geminin-GFP, except that immobilization was based on anti-Flag antibodies. n = 30–40 cell units.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated antiGFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Labeling, Ubiquitin Proteomics, SDS Page, Autoradiography, Incubation, Control, Variant Assay, Mutagenesis, Comparison

Journal: Communications biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts.

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: Fig. 4 Method sensitivity. a Time-dependent degradation assay of 35S-labeled Flag-Securin-GFP in NDB mitotic extracts. Assays were performed with undiluted IVT product (100%) or following 4x/10x dilution in reticulocyte lysate (25 and 10%, respectively). In all assays, 1 µl substrate was incubated in 20 µl cell extracts. Samples were snap-frozen in 20 min intervals and assayed by SDS-PAGE and autoradiography. b Equivalent degradation assays performed with non-radioactive Flag-Securin-GFP. Time-point samples were loaded on the chip for immobilization (via anti-GFP-biotinylated antibody) and detection (GFP fluorescence). The plots summarize data from three experiments, 15–20 cell units per experiment. Normalized mean and standard error values are shown (left). Representative raw data are shown on the right.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated antiGFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Degradation Assay, Labeling, Incubation, SDS Page, Autoradiography

Journal: Communications biology

Article Title: Affinity microfluidics enables high-throughput protein degradation analysis in cell-free extracts.

doi: 10.1038/s42003-022-04103-3

Figure Lengend Snippet: Fig. 5 Protein degradation on chip. a, b Schematic illustration of complete on-chip degradation assay by pDOC. IVT protein products are immobilized to the surface of the ‘protein chamber’ via anti GFP biotinylated antibody (see more information in Fig. 1). The closing of the MITOMI button valve traps the protein. All remains are washed away with PBS. Proper expression and immobilization of the target proteins are validated by scanning. Next, cell-free extracts are loaded into the extract chamber and trapped by closing the neck valve. The reaction begins with the opening of neck and MITOMI button valves, which allows diffusion of cell extracts into the ‘protein chamber’ and mixing with the target protein. The chip is placed on a 30 °C hot plate and scanned at 15 min intervals to provide kinetic information in real time. c GFP-tagged Securin and p27 IVT products were immobilized via anti-GFP- biotinylated antibody. Extract chambers were then filled with NDB mitotic extracts that support ubiquitination of Securin, but not of its non-degradable variant (Δ64) and p27 (negative control validating assay specificity). Protein degradation was assayed for 1 h during which the chip was scanned five times. The plot depicts mean GFP signals normalized to t0 and standard error bars; n = 14–30 cell units. Representative raw data for p27 and Securin are shown on the right.

Article Snippet: Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated antiGFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 μg/μl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied.

Techniques: Degradation Assay, Expressing, Diffusion-based Assay, Ubiquitin Proteomics, Variant Assay, Negative Control

Journal: PLoS ONE

Article Title: Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

doi: 10.1371/journal.pone.0115630

Figure Lengend Snippet: a) Fluorescent microarray scanner measuring relative abundance of initiator prior to polymerization labeling. Scale bar = 1 mm. b) Relative initiator concentration on surface for spots printed from the indicated concentration of biotinylated-BSA and reacted with the SA-initiator complex. Measurements based on initiator fluorescence (ex/em = 525/545 nm). c) Thickness of spots of indicated Bio-BSA concentration. Data are mean ± standard deviation. n = 16.

Article Snippet: Biotinylated polyclonal goat IgG anti-mouse IgG (H+L; catalogue #BA-9200) and Vectashield hardset mounting medium was purchased from Vector Laboratories (Burlingame, CA).

Techniques: Microarray, Labeling, Concentration Assay, Fluorescence, Standard Deviation

Journal: PLoS ONE

Article Title: The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

doi: 10.1371/journal.pone.0021269

Figure Lengend Snippet: A) TIGR4 bacteria were labeled with anti-RrgA, RrgB clade I, RrgC, BgaA (beta-galactosidase), PhtA (pneumococcal histidine triad protein A) or PspC (pneumococcal surface protein C, also known as CbpA,) primary antibodies (1∶400 dilution), and with FITC anti-mouse IgG secondary antibodies (1∶100 dilution). Bacterial staining was analyzed by flow cytometry (FACS-Calibur). Sera of mice immunized with PBS were used as negative control. B,C) TIGR4 bacteria were processed for immunofluorescence, stained with mouse anti-RrgB antibodies (1∶2000 dilution) (red) and with S. pneumoniae anti-capsular antibodies (Omniserum 1∶2000 dilution) (green). Imaging was performed with a confocal microscope. Scale bar is 4 µm in panel B and 1 µm in panel C.

Article Snippet: TIGR4 low pilus expressing bacteria were incubated with rabbit anti-RrgB antibodies (1∶400 dilution) and then with goat anti-rabbit IgG biotin conjugated (Abcam, 1∶1000 dilution) antibodies.

Techniques: Labeling, Staining, Flow Cytometry, Negative Control, Immunofluorescence, Imaging, Microscopy

Journal: PLoS ONE

Article Title: The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

doi: 10.1371/journal.pone.0021269

Figure Lengend Snippet: Bacteria containing either PI-1 clade I (A), clade II (B) or clade III (C), were labeled with clade specific anti-RrgB antibodies (1∶400 dilution) and FITC anti-mouse IgG secondary antibodies (1∶100 dilution). Pilus-1 expression was then analyzed by flow cytometry (FACS-Calibur).

Article Snippet: TIGR4 low pilus expressing bacteria were incubated with rabbit anti-RrgB antibodies (1∶400 dilution) and then with goat anti-rabbit IgG biotin conjugated (Abcam, 1∶1000 dilution) antibodies.

Techniques: Labeling, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

doi: 10.1371/journal.pone.0021269

Figure Lengend Snippet: A) High and low pilus expressing sub-populations of strains TIGR4 (Clade I), 19F Taiwan 14 (Clade I), OREP4 (Clade I), 6B Finland 14 (Clade II) and 35B SME 15 (Clade III) were labeled with clade specific anti-RrgB antibodies (1∶400 dilution) and FITC anti-mouse IgG secondary antibodies (1∶100 dilution). Pilus-1 expression was then analyzed by flow cytometry (FACS-Calibur). B) Schematic representation of PI-1. C) Log 2 ratio values indicating the PI-1 genes differential expression in High vs. Low pilus expressing sub-populations in the five above mentioned strains, as measured by spotted DNA microarray analysis. The data are measures of relative gene expression during in vitro growth in liquid cultures. The values reported for each gene are the mean of all the spots and their replicates within the array and of two independent experiments (bars represent standard deviations). D) Absolute gene expression levels of PI-1 genes measured for TIGR4 high and low pilus expressing sub-populations by microarray hybridization. Absolute expression levels reported for each gene are the mean of all the spots and their replicates within the array and of two independent experiments (bars represent the obtained standard deviations).

Article Snippet: TIGR4 low pilus expressing bacteria were incubated with rabbit anti-RrgB antibodies (1∶400 dilution) and then with goat anti-rabbit IgG biotin conjugated (Abcam, 1∶1000 dilution) antibodies.

Techniques: Expressing, Labeling, Flow Cytometry, Microarray, In Vitro, Hybridization