Journal: Journal of Biological Chemistry

Article Title: Heterodimerization of α2A- and β1-Adrenergic Receptors

doi: 10.1074/jbc.m207968200

Figure Lengend Snippet: FIG. 2. Co-internalization of 1AR with 2AAR. HA-2AAR and FLAG-1AR were expressed either separately (solid bars) or together (striped bars) in HEK-293 cells. The internalization of 2AAR (A) and 1AR (B) was examined using a luminometer-based assay following 10-min stimulations with the -adrenergic agonist isoproterenol (Iso; 10 m), the 2-adrenergic agonist UK 14,304 (UK; 10 M), or a combina- tion of the two agonists together. As shown in A, 2AAR exhibited 15% internalization in response to UK stimulation but no significant inter- nalization in response to isoproterenol under any condition. Conversely, as shown in B, 1AR exhibited 25–30% internalization in response to isoproterenol but also exhibited 15% internalization in response to stimulation with UK. This effect was only observed, however, when 2AAR was coexpressed (** indicates significantly different from 1AR alone, p 0.01). These data suggest that 1AR can co-internalize with agonist-activated 2AAR. The bars and error bars represent the means S.E. for 4–5 independent experiments for each condition, with each experiment being performed in triplicate.

Article Snippet: After another 48 h, cells were treated with varying concentrations of isoproterenol for 10 min and harvested with cell harvest buffer (50 mM Tris, pH 7.4, 250 M Ro 20-1724 (Tocris, Ellisville, NJ), 5 mM MgCl2, 1 mM ATP, and 1 M GTP).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Heterodimerization of α2A- and β1-Adrenergic Receptors

doi: 10.1074/jbc.m207968200

Figure Lengend Snippet: FIG. 3. Immunofluorescence confocal microscopy reveals ago- nist-promoted co-internalization of 2A- and 1-adrenergic re- ceptors. HA-2AAR (red) and FLAG-1AR (green) were co-transfected into HEK-293 cells and visualized using secondary antibodies coupled to rhodamine and FITC, respectively. In the absence of agonist stimu- lation, immunostaining for both receptors was found predominantly in the plasma membrane (A–C). Stimulation with isoproterenol (Iso) for 10 min induced significant mobilization of 1AR inside the cell (D) but had no significant effect on the subcellular distribution of 2AAR (E and F). Stimulation with UK 14,034 (UK), in contrast, resulted in significant internalization of both 2AAR (H) and 1AR (G) and marked co-local- ization of the two receptors in intracellular regions (I, with co-localiza- tion indicated in yellow). The specificity of staining was determined in control (Con) experiments using both untransfected and transfected cells incubated in the absence and presence of the relevant primary antibodies. These data are representative of 3–5 experiments for each condition.

Article Snippet: After another 48 h, cells were treated with varying concentrations of isoproterenol for 10 min and harvested with cell harvest buffer (50 mM Tris, pH 7.4, 250 M Ro 20-1724 (Tocris, Ellisville, NJ), 5 mM MgCl2, 1 mM ATP, and 1 M GTP).

Techniques: Immunofluorescence, Confocal Microscopy, Transfection, Immunostaining, Clinical Proteomics, Membrane, Staining, Control, Incubation

Journal: Journal of Biological Chemistry

Article Title: Heterodimerization of α2A- and β1-Adrenergic Receptors

doi: 10.1074/jbc.m207968200

Figure Lengend Snippet: FIG. 5. Coexpression of 1AR with 2AAR alters the potency of isoproterenol-induced stimulation of adenylyl cyclase. HEK-293 cells were transfected with either 1AR alone (filled squares, solid line) or 1AR/2AAR (open triangles, dotted line). Expression levels of the 1AR were identical for the two transfection conditions, as assessed by Western blot. The cells were stimulated with increasing concentrations of isoproterenol, and agonist-induced rises in cellular cyclic AMP were quantified. The maximal extent of cyclic AMP produced in the 1AR/2AAR cells was 106 8% of that produced in the cells trans- fected with only 1AR. The EC50 for isoproterenol stimulation of 1AR alone was 0.16 0.02 nM, as compared with 0.68 0.17 for 1AR/ 2AAR (significantly different from 1AR alone, p 0.01). The points and error bars represent the means and S.E. values for four independ- ent determinations.

Article Snippet: After another 48 h, cells were treated with varying concentrations of isoproterenol for 10 min and harvested with cell harvest buffer (50 mM Tris, pH 7.4, 250 M Ro 20-1724 (Tocris, Ellisville, NJ), 5 mM MgCl2, 1 mM ATP, and 1 M GTP).

Techniques: Transfection, Expressing, Western Blot, Produced

Journal: Molecular Cell

Article Title: DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

doi: 10.1016/j.molcel.2020.05.035

Figure Lengend Snippet: DDI2 KO Cells Accumulate Slow-Migrating Ubiquitylated Proteins (A) DDI2 western blot analysis of MRC5VA WT and DDI2 KO (Δ) cells. Asterisks denote non-specific bands. (B) Western blot analysis of ubiquitylated proteins after DSK2 chromatography of extracts from WT and DDI2 KO (Δ) cells. For ease of comparison between WT and Δ, increasing amounts of protein was loaded. See quantification in A–S1C. (C) As in (B), but using another SDS-PAGE gel type, as indicated. (D) Stability of ubiquitylated protein species in WT and DDI2 KO (Δ) extracts after cycloheximide inhibition of new protein production at time = 0. Gel as in (B). See quantification in D and S1E. (E) As in (A), but in U266B cells. (F) Incucyte Live-Cell Analysis of U266B multiple myeloma re-growth after a 16 h treatment with 5 nM PS-341 (Bortezomib) and control (DMSO). Confluence analyzed in Prism. Experiments were performed in triplicate, and numbers represent mean ± SD. See Figure S2 for MRC5VA cells. (G) Western blot analysis of ubiquitylated proteins after treatment of U266B multiple myeloma cells with different concentrations of Bortezomib/PS-341. Cell extracts were analyzed directly, on 4%–15% Criterion TGX gels. See also and .

Article Snippet: Approximately, 50 μg protein/lane was separated on 4%–12% Bis-Tris Criterion XT (BioRad 3450124/5), 3%–8% Tris-Acetate Criterion XT (BioRad, 3450130), or 4%–15% TGX gels (BioRad, 56711084/5), and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, 10600002, 10600019).

Techniques: Western Blot, Chromatography, Comparison, SDS Page, Inhibition, Cell Analysis, Control

Journal: Molecular Cell

Article Title: DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

doi: 10.1016/j.molcel.2020.05.035

Figure Lengend Snippet: Purified DDI Proteins Digest the Slowly Migrating Ubiquitylated Species from DDI2 KO Cells (A) Western blot analysis of ubiquitylated proteins after incubation of DDI2 KO extract with the indicated DDI proteins (human DDI1 and DDI2 from HEK293; HRAD23 (R23) from Mybiosource; Leishmania major DDI1 (L.m.) from insect cells; see Figure S3 for purified proteins) (3%–8% Tricine gels). The extent of the effect of RAD23 varies between experiments, but it never has activity on its own. (B) As in (A), but testing the effect of proteasome inhibitor MG132 (4%–15% TGX gel). Right panel is a stronger exposure of the area indicated on the left. Numerous iterations of this experiment have been performed, both in extracts and with purified ubiquitylated proteins, and proteasome inhibition consistently has no effect. See Figure S4 for quantification. (C) As in (A), but testing recombinant DDI2 proteins, all derived from E. coli , including DDI2 D→N (4%–15% TGX gel). Note that untagged (lane 3) and His-SUMO-tagged (lane 4) WT DDI2 are equally active. (D) As in (C), but using DSK2-purified ubiquitylated substrates. The lower panel is a stronger exposure. Only the top of the gel is shown. See also quantification of an independent experiment in A–S5C. (E) As in (D), but including Ulp1 digestion, and probing the same membrane with anti-ubiquitin and anti-SUMO antibodies, as indicated (4%–12% Bis-Tris gel). The panel on the right is an enlargement of the area indicated on the left. See also .

Article Snippet: Approximately, 50 μg protein/lane was separated on 4%–12% Bis-Tris Criterion XT (BioRad 3450124/5), 3%–8% Tris-Acetate Criterion XT (BioRad, 3450130), or 4%–15% TGX gels (BioRad, 56711084/5), and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, 10600002, 10600019).

Techniques: Purification, Western Blot, Incubation, Activity Assay, Inhibition, Recombinant, Derivative Assay, Membrane, Ubiquitin Proteomics

Journal: Molecular Cell

Article Title: DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

doi: 10.1016/j.molcel.2020.05.035

Figure Lengend Snippet: DDI2 Cleaves Ubiquitylated NRF1 Protein In Vitro (A) Western blot analysis of NRF1 protein, isolated via GST-DSK2 chromatography, after treatment with DDI2, RAD23, and USP2, as indicated (4%–15% TGX). The mobility of NRF1 and cleaved NRF1 is shown on the right (see also arrow for cleaved NRF1). Zoomed-in images of lanes 13 and 14 and 19 and 20 are shown below. Note that large amounts of extract were used as input for GST-DSK2 purification, meaning that in relative terms, ~20× “extract equivalents” were loaded in lanes 5–20 compared to lanes 1–4. See also Figure S6 A. (B) Reprobing of the membrane from (A), with anti-ubiquitin antibodies. For simplicity, only the relevant lanes 5–9 are shown. (C) Experiment as in (A), but testing DDI2 D→N , as indicated. (D) As in (A) and (B), but testing cleavage of exogenously expressed, Myc-tagged versions of NRF1. N, normal NRF1 sequence. m, mutated sequence. Lower panel, lanes 2 and 6 are shown in the enlargement. The “dot” in lane 6 is not a cleavage band. See also independent experiment in Figure S6 B.

Article Snippet: Approximately, 50 μg protein/lane was separated on 4%–12% Bis-Tris Criterion XT (BioRad 3450124/5), 3%–8% Tris-Acetate Criterion XT (BioRad, 3450130), or 4%–15% TGX gels (BioRad, 56711084/5), and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, 10600002, 10600019).

Techniques: In Vitro, Western Blot, Isolation, Chromatography, Purification, Membrane, Ubiquitin Proteomics, Sequencing

Journal: Molecular Cell

Article Title: DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

doi: 10.1016/j.molcel.2020.05.035

Figure Lengend Snippet:

Article Snippet: Approximately, 50 μg protein/lane was separated on 4%–12% Bis-Tris Criterion XT (BioRad 3450124/5), 3%–8% Tris-Acetate Criterion XT (BioRad, 3450130), or 4%–15% TGX gels (BioRad, 56711084/5), and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, 10600002, 10600019).

Techniques: Virus, Recombinant, Multiplex sample analysis, Ubiquitin Proteomics, Membrane, Mass Spectrometry, Western Blot, Sequencing, Plasmid Preparation, Software, Transfection

Journal: Veterinary Research

Article Title: Swine influenza A virus infection sets the local immunological landscape in subsequent infection with porcine reproductive and respiratory syndrome virus

doi: 10.1186/s13567-025-01536-6

Figure Lengend Snippet: Clinical signs and lung lesions. A Rectal temperature. On D0, the red arrow indicates swIAV inoculation, and on D7, the blue arrow indicates PRRSV inoculation. Statistical analysis was performed using the Kruskal–Wallis unpaired, non-parametric test. Different letters (a-d) indicate that the considered group (specified by its color) was significantly different from the Mock group (a), from the swIAV group (b), from the PRRSV group (c) or from the swIAV/PRRSV group (d) with p < 0.05 (mean ± SD; n = 5–10). B Average daily weight gain (mean; n = 5–10). Statistical analysis was performed using the Kruskal–Wallis unpaired, non-parametric test, (*) p < 0.05. C , D Lung sections were evaluated for histopathological lesions. C Jung score and D Composite score (mean; n = 5). Statistical analysis was performed using the Mann–Whitney unpaired, non-parametric test, (*) p < 0.05 or (**) p < 0.01.

Article Snippet: The swIAV strain A/swine/Ille et Vilaine/0415/2011 (H1 hu N2, HA-clade 1B.1.2.3) and the so-called “Finistère” PRRSV strain (referenced as PRRS-FR-2005-29-24-1; PRRSV-1 subtype 1), both isolated from pig farms in Brittany, France, are part of the collections of the French Agency for Food, Environmental, and Occupational Health & Safety (ANSES, Ploufragan, France).

Techniques: MANN-WHITNEY

Journal: Veterinary Research

Article Title: Swine influenza A virus infection sets the local immunological landscape in subsequent infection with porcine reproductive and respiratory syndrome virus

doi: 10.1186/s13567-025-01536-6

Figure Lengend Snippet: Principal component analysis of swIAV/PRRSV and PRRSV infected groups. PCA includes PRRSV loads in the lung (Figure B) along with all the other variables depicted in Figures , , , , encompassing the data from PRRSV-infected and swIAV/PRRSV-infected animals (D10 PRRSV, D10 swIAV/PRRSV, D14 PRRSV, D14 swIAV/PRRSV groups). The first two PCA dimensions (Dim) accounted for 29% (Dim1) and 18% (Dim2) of the data complexity respectively. A Graph of individuals, each symbol depicts one animal from PRRSV groups (blue triangles) or swIAV/PRRSV groups (green lozenges). Confidence ellipses define the regions that contains 95% of the samples from each condition (PRRSV or swIAV/PRRSV) that can be drawn from the underlying Gaussian distribution. B Projections of the 22 variables (Composit score, Young score; PRRSV Lung load; serum IL-1α, IL-1β, IL-6, IL-8, IL-12, IL-18 concentrations; BALC content in AM, moDC, cDC1, cDC2, Tconv, Treg, TICOSKi67, TregICOSKi67, Cl.6, Cl.7, Cl.8, Cl.9, Cl.10) on the two first dimensions.

Article Snippet: The swIAV strain A/swine/Ille et Vilaine/0415/2011 (H1 hu N2, HA-clade 1B.1.2.3) and the so-called “Finistère” PRRSV strain (referenced as PRRS-FR-2005-29-24-1; PRRSV-1 subtype 1), both isolated from pig farms in Brittany, France, are part of the collections of the French Agency for Food, Environmental, and Occupational Health & Safety (ANSES, Ploufragan, France).

Techniques: Infection