Journal: EMBO Molecular Medicine

Article Title: Protein-based tools for the detection and characterisation of Oropouche virus infection

doi: 10.1038/s44321-025-00291-7

Figure Lengend Snippet: ( A ) Amino acid sequence alignment of selected nanobodies that recognise OROV N (NpXX) or Gc spike (SpXX). Complementarity Determining Regions (CDRs) as defined by IGMT (Giudicelli et al, ) are highlighted. ( B ) Coomassie-stained SDS-PAGE of nanobodies that had been biotinylated in vitro. Upper bands represent the Avi-tagged, biotinylated nanobodies while lower bands represent nanobodies where the tag had been lost, presumably by proteolysis during the purification procedure. ( C ) Electrophoretic mobility shift assay to confirm biotinylation. After boiling of the nanobody in SDS-PAGE loading buffer, streptavidin was added at a 2:1, 1:1 or 1:2 molar ratio (nanobody:streptavidin) and samples were subjected to SDS-PAGE. Appearance of a high apparent molecular weight band, and disappearance of the low molecular weight band, confirms biotinylation of the nanobody. Asterisk (*) denotes streptavidin that was boiled before SDS-PAGE, rather than being added to the sample buffer after boiling. ( D ) Coomassie-stained SDS-PAGE of His 8 -tagged nanobodies. ( E ) OROV N competition BLI sensorgrams. Streptavidin biosensors loaded with bNbs were sequentially incubated with 1 µM OROV N then with 1 µM OROV N plus 25 µM of each competitive nanobody, with biosensor regeneration between incubation (association) cycles. After the final competition step, the biosensor was incubated with 1 µM OROV N to confirm that the bNbs remained active. For each association in the presence of competitive nanobodies, the response for each sensor at 180 s (dotted grey line) was divided by the response of the same sensor in the presence of OROV N alone at 180 s to generate the heatmap shown in Fig. . .

Article Snippet: Plates were incubated with biotinylated nanobodies (2 μg/mL in blocking buffer) for 1 h, HRP-conjugated streptavidin (1:10,000 dilution, Thermo Fisher Scientific N100) for 15 min, and then developed.

Techniques: Sequencing, Staining, SDS Page, In Vitro, Purification, Electrophoretic Mobility Shift Assay, Molecular Weight, Incubation

Journal: EMBO Molecular Medicine

Article Title: Protein-based tools for the detection and characterisation of Oropouche virus infection

doi: 10.1038/s44321-025-00291-7

Figure Lengend Snippet: ( A ) Nanobody detection of purified OROV antigens. Antigens were immobilised on the capture surface via passive adsorption (2 µg/mL) and detected via incubation with specified biotinylated nanobodies (bNbs) plus streptavidin-HRP. CRIV Gc spike was included as a negative control. The data represent one experiment. ( B ) Sandwich ELISA using purified OROV antigens to determine nanobody competition groups. Surfaces were coated with nanobodies (2 µg/mL) before being incubated with antigen then detection bNbs and streptavidin-HRP. For non-competing nanobodies, data are representative of two independent experiments. For competing Gc spike nanobodies the experiment was performed once. ( C ) Competition biolayer interferometry (BLI) to map nanobody competition for OROV N epitopes. Streptavidin biosensors were loaded with bNbs then incubated with 1 µM OROV N plus 25 µM competitor nanobody. Heatmap shows BLI responses after 180 s of association, normalised relative to the response obtained for each bNb-loaded biosensor incubated with 1 µM OROV N alone. BLI traces are shown in Fig. . Data are representative of two independent experiments. ( D ) Limit of detection (LOD) of purified Gc spike (left) or N (right) in sandwich ELISA using optimised nanobody pairings. LOD is calculated as (3.3 * standard deviation [background])/slope. Data are representative of two independent experiments. ( E ) Detection of Gc spike (left) and N (right) in laboratory stocks of OROV strains TRVL (Anderson et al, ) and BeAn19991 (Acrani et al, ; Aquino et al, ) by sandwich ELISA. Data shown are from one experiment performed in technical triplicate. .

Article Snippet: Plates were incubated with biotinylated nanobodies (2 μg/mL in blocking buffer) for 1 h, HRP-conjugated streptavidin (1:10,000 dilution, Thermo Fisher Scientific N100) for 15 min, and then developed.

Techniques: Purification, Adsorption, Incubation, Negative Control, Sandwich ELISA, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Protein-based tools for the detection and characterisation of Oropouche virus infection

doi: 10.1038/s44321-025-00291-7

Figure Lengend Snippet: ( A , B ) Coomassie-stained SDS-PAGE of ( A ) untagged nanobody-Fc fusions, and ( B ) biotinylated nanobody-Fc fusions. ( C ) Electrophoretic mobility shift assay to confirm biotinylation. After boiling of the nanobody-Fc fusion in SDS-PAGE loading buffer, streptavidin was added at a 8:1, 4:1, 2:1, 1:1 or 1:2 molar ratio (nanobody-Fc fusion:streptavidin) and samples were subjected to SDS-PAGE. Appearance of a high apparent molecular weight bands, and reduction of the lower molecular weight band, confirms biotinylation of the nanobody-Fc fusion.

Article Snippet: Plates were incubated with biotinylated nanobodies (2 μg/mL in blocking buffer) for 1 h, HRP-conjugated streptavidin (1:10,000 dilution, Thermo Fisher Scientific N100) for 15 min, and then developed.

Techniques: Staining, SDS Page, Electrophoretic Mobility Shift Assay, Molecular Weight

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 1. Western blot analysis of CFTR and STAT1 expression in un- treated (lane 1), PMA-treated (lane 2), TNF-treated (lane 3), and IFN- treated (lane 4) T84 (A) and RCMC (B). T84 cells were treated with PMA (10 ng/ml), human recombinant TNF (10 ng/ml), or IFN (10 ng/ml) for 24 h. RCMC were treated with PMA (10 ng/ml) or rat recombinant TNF (10 ng/ml) or IFN (10 ng/ml) for 24 h. Cell lysates were resolved on an 4 to 12% SDS-PAGE and blotted with anti-CFTR, anti-STAT1, or anti-actin antibody. Western blots shown represent three independent experiments. The effect of 24 h IFN treatment (80 ng/ml) on CFTR expression was characterized in T84 cells (C) and rat peritoneal mast cells (D), using confocal laser scanning microscopy (bar on the figure represents 10 m).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Western Blot, Expressing, Recombinant, SDS Page, Confocal Laser Scanning Microscopy

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 4. A, IFN-mediated up-regulation of mast cell CFTR protein requires activation of JAK2 and/or p38 and ERK. RCMC, LAD2, and T84 were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SB202190; 30 mg/ml), or ERK MAPK inhibitor (U0126; 30 mg/ml) for 24 h. Representative of three inde- pendent experiments. B, densitometry summary of data in Fig. 4. RCMC were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SP202190; 30 mg/ml), or ERK inhibitor (U0126; 30 mg/ml) for 24 h, and expression of CFTR (black columns) and STAT1 (gray columns) was analyzed by Western blot (n 3 independent experiments; error bars represent S.E.M.). As- terisks represent statistical significance as determined by Student’s t test (p 0.05).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Western Blot

Journal:

Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27 -Haploinsufficient Mammary Epithelial Cells but Impaired in p27 -Null Cells

doi: 10.1128/MCB.22.7.2204-2219.2002

Figure Lengend Snippet: Overexpression of ErbB2 increases cyclin D1 expression in p27+/+ and p27+/− PMECs but not p27−/− PMECs. (A) Western analysis of whole-cell extracts (WCE), 9E10 immunoprecipitates (IP: 9E10), or ErbB2 immunoprecipitates (IP: ErbB2) from uninfected p27+/+ PMECs (NI) or p27+/+ PMECs infected with pBabe or pBabe-erbB2. Blots were probed with antibodies against ErbB2 (left panels) or phosphotyrosine (right panel). (B) PMECs infected with empty pBabe or pBabe-erbB2 were cultured for 10 days from a single-cell suspension embedded in growth factor-reduced Matrigel. Cultures were photographed at magnifications of ×100 (panels 1 to 6) and ×400 (panels 7 to 12). The photographs shown are representative of results obtained in three independent experiments. Panels 10 to 12, whole-mount immunohistochemical detection of E-cadherin in organoid cultures. DAPI-stained nuclei are shown to the right. (C) Western analysis of cell extracts harvested from PMEC monolayers infected with pBabe, pBabe-erbB2, pBabe-cyclin D1, or pBabe-cyclin D1(T286A). WB, primary antibodies used for Western blot analysis, listed at the left. The 9E10 antibody is against the myc epitope tag. Molecular masses are shown at right in kilodaltons. The results presented here are representative of results obtained in three independent experiments. (D) Western analysis of cell extracts harvested from PMEC organoid cultures infected with pBabe, pBabe-erbB2, pBabe-cyclin D1, or pBabe-cyclin D1(T286A). (E) Northern analysis of total cellular RNA harvested from infected PMECs. The cDNA probe used for hybridization is indicated (cyclin D1 and GAPDH). The positions of the 28S and 18S rRNAs are indicated.

Article Snippet: Slides were incubated with a cyclin D1 polyclonal antibody (diluted 1:100 in PBS) (Santa Cruz Biotechnology) for 1 h at room temperature, followed by incubation with an anti-rabbit antibody conjugated to Cy3 fluorochrome (diluted 1:2,500 in PBS) (Molecular Probes, Inc.).

Techniques: Over Expression, Expressing, Western Blot, Infection, Cell Culture, Immunohistochemical staining, Staining, Northern Blot, Hybridization

Journal:

Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27 -Haploinsufficient Mammary Epithelial Cells but Impaired in p27 -Null Cells

doi: 10.1128/MCB.22.7.2204-2219.2002

Figure Lengend Snippet: Nuclear localization of cyclin D1 is impaired in p27−/− PMECs. (A) Western analysis of cytoplasmic and nuclear extracts from infected PMECs with antibodies against cyclin D1, p27, and PCNA. (B) Immunofluorescence analysis was used to detect cellular localization of cyclin D1 in infected PMECs. DAPI staining of nuclei is pictured directly below the corresponding cyclin D1 immunofluorescence. Magnification, ×400. Values shown represent the percentage of total nuclei that were positive for cyclin D1 staining. A total of 500 nuclei were counted per experimental condition.

Article Snippet: Slides were incubated with a cyclin D1 polyclonal antibody (diluted 1:100 in PBS) (Santa Cruz Biotechnology) for 1 h at room temperature, followed by incubation with an anti-rabbit antibody conjugated to Cy3 fluorochrome (diluted 1:2,500 in PBS) (Molecular Probes, Inc.).

Techniques: Western Blot, Infection, Immunofluorescence, Staining

Journal:

Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27 -Haploinsufficient Mammary Epithelial Cells but Impaired in p27 -Null Cells

doi: 10.1128/MCB.22.7.2204-2219.2002

Figure Lengend Snippet: Activity of Cdk4 is impaired in p27−/− PMECs and is not restored by ErbB2 or cyclin D1 overexpression. (A) Cell extracts from infected PMECs were immunoprecipitated (IP) with an antibody against Cdk4. Immune complexes were divided in half and tested in an in vitro kinase assay with pRb as a substrate (upper panel) or used for Western blot analysis (WB) with a Cdk4 antibody (lower panel). Kinase reactions were performed in the presence of [γ-32P]ATP and then resolved by SDS-PAGE. (B) Whole-cell extracts from infected PMECs were subjected to Western blot analysis using the antibodies indicated at the right. (C) Whole-cell extracts from PMECs infected with pBabe-p27 or pBabe-E2F1 or from uninfected PMECs (N.I.) were immunoprecipitated with an antibody against Cdk4. Products were divided in half and used in an in vitro kinase reaction against pRb or in Western analysis for Cdk4 as described in panel A. Whole-cell extracts were used for Western analysis with p27 or E2F1 antibodies. (D) p27−/− PMECs infected with the indicated viruses were cultured in the presence of cycloheximide (Cyclohex) (1 μg/ml). Cell extracts were analyzed for cyclin D1 expression at various time points following cycloheximide administration. (E) Whole-cell extracts from p27−/− PMECs infected with the indicated viruses or from uninfected p27−/− PMECs (N.I.) were used for detection of cyclin D1 by Western analysis (top panel) or were immunoprecipitated with an antibody against Cdk4 and analyzed for coprecipitation of cyclin D1.

Article Snippet: Slides were incubated with a cyclin D1 polyclonal antibody (diluted 1:100 in PBS) (Santa Cruz Biotechnology) for 1 h at room temperature, followed by incubation with an anti-rabbit antibody conjugated to Cy3 fluorochrome (diluted 1:2,500 in PBS) (Molecular Probes, Inc.).

Techniques: Activity Assay, Over Expression, Infection, Immunoprecipitation, In Vitro, Kinase Assay, Western Blot, SDS Page, Cell Culture, Expressing