Journal: Blood Coagulation & Fibrinolysis

Article Title: Performance evaluation of Stago sthemO 301 hemostasis analyzer for routine testing, coagulation factors, and inhibitors

doi: 10.1097/MBC.0000000000001421

Figure Lengend Snippet: Passing-Bablok regression plots with data of STAR Max (predicate device) on the x -axis and data of sthemO 301 on the y -axis. Black lines represent the identity lines and light grey lines the reference lines. (a) Routine coagulation parameters. (b) Coagulation factors assays. (c) D-dimer and coagulation inhibitors assays. Ag, antigen; aPTT, activated partial thrombin time; AT, antithrombin activity; Chrom, chromogenic; PC, protein C activity; PS, protein S activity; PT, prothrombin time; s, seconds; TT, thrombin time.

Article Snippet: PC Chrom (IU/dl) , STA-Stachrom Protein C , Chromogenic , sthemO PC chrom , Chromogenic.

Techniques: Coagulation, Activity Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Protease-activated receptor 1 as an endogenous model of peptidergic Gαq-Gα12-biased G protein signaling

doi: 10.3389/fmolb.2026.1822420

Figure Lengend Snippet: Activated protein C (aPC) exhibits PAR1 coupling to only Gα12. (A) Heatmap of EC 50 values for the 13 individual Gα subunits determined from TRUPATH concentration-response fits. The map displays EC 50 values for the reference receptors included in the study, along with PAR1 responses measured using activated protein C (aPC). Each cell corresponds to the EC 50 (M) calculated from the concentration-response curve for the indicated Gα. Color intensity denotes potency. Grey boxes indicate Gα/receptor pairs for which no response was observed (NR). White boxes denote pairs for which coupling was not determined (ND). (B) Representative TRUPATH concentration-response curves for Gαq (left) and Gα12 (right). Curves compare NTSR1 stimulation with neurotensin and PAR1 stimulation with aPC. Symbols show mean ± SEM across biological replicates; curves are nonlinear fits to the pooled replicate data used to extract EC 50 values. (C) TGFα-shedding assay percentage of released AP-TGFα normalized to baseline, showing dose-dependent responses of PAR1 with aPC in the presence or absence of FR900359 . NTSR1 with neurotensin serves as the reference. Data are shown as the mean signal ±SEM. (D) PRESTO-Tango β-arrestin-2 recruitment assay comparing fold change of DRD2 stimulation with dopamine and PAR1 stimulation with aPC. Data are plotted as mean ± SEM. All plotted points represent the mean of three independent biological replicates, each measured in quadruplicate (N = 3, n = 4).

Article Snippet: Activated Protein C (aPC) was purchased from Enzyme Research Laboratories (Cat# APC).

Techniques: Concentration Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Protease-activated receptor 1 as an endogenous model of peptidergic Gαq-Gα12-biased G protein signaling

doi: 10.3389/fmolb.2026.1822420

Figure Lengend Snippet: TRE-MPRA dataset nominates PAR1-responsive transcriptional elements, with validation revealing Gαq-dependent NFκB1 and Gαq-independent THRB reporters. (A) Volcano plot of differential TRE activity in PAR1-expressing HEK293 cells stimulated with thrombin versus vehicle. Each point represents an individual TRE. Light blue points mark TREs belonging to the highest-responding promoter architecture used for downstream analysis, light gray points are TREs from all other architectures, and dark gray points are negative-control TREs. Red points highlight the top 10 thrombin-responsive TREs within the highest-responding architecture. (B) Summary table of the top 10 TREs, including element identity, promoter architecture, and differential activity statistics. (C) Individual luciferase validation of the NFκB1 TRE in HEK293 cells expressing PAR1, stimulated with thrombin or activated protein C (aPC) in the presence or absence of the Gαq-family inhibitor FR900359 . NFκB1 activity is robustly induced by thrombin, abolished by FR900359 , and not detectably induced by aPC. (D) Individual luciferase validation of the THRB TRE under the same conditions. Both thrombin and aPC induce THRB reporter activity, and THRB is not suppressed by FR900359 . Data are plotted as mean ± SEM. All plotted points represent the mean of three independent biological replicates, each measured in quadruplicate (N = 3, n = 4).

Article Snippet: Activated Protein C (aPC) was purchased from Enzyme Research Laboratories (Cat# APC).

Techniques: Biomarker Discovery, Activity Assay, Expressing, Negative Control, Luciferase

Journal: Journal of Nanobiotechnology

Article Title: A versatile nanobody platform for live and super-resolution imaging of synaptic vesicle dynamics and plasticity in rodent and human neurons

doi: 10.1186/s12951-026-04489-w

Figure Lengend Snippet: Syt1 is found in close proximity to the ciliary neurotrophic factor receptor (Cntfr) and regulates synaptic vesicle dynamics. a-c ) NbLumSyt1-APEX2 allows efficient biotinylation of proteins in the proximity of Syt1 upon live uptake in hippocampal neurons to facilitate live-cell proteomic mapping. Representative images of neurons upon uptake of NbLumSyt1-APEX2, where biotinylated proteins are revealed with fluorescent streptavidin (a) . In the absence of H 2 O 2 , only a few endogenous biotinylated proteins are observable. In the presence of all the components, the reaction occurred efficiently, as revealed in western blot analysis of labeled neurons ( b ). To identify the interactors of Syt1, in situ proximity labeling was performed with NbLumSyt1-APEX2 ( c ). The electron microscopy image in the scheme is an example of the labeled vesicles, as revealed upon photoconverting 3,3’-diaminobenzidine (DAB) into a stable, electron microscopically visible dark product. d ) Protein intensities measured with LC‒MS/MS at the input and upon enrichment of the biotinylated proteins. Two controls were used: neurons without nanobodies or neurons where an unrelated nanobody (anti-ALFA-Nb) was provided in the medium. Note that since the primary neurons do not express the ALFA tag, this control will reveal the effect of the unspecific biotinylation of the membranes occurring during the labeling period. Note that Syt1, as expected, is efficiently biotinylated and enriched upon IP with streptavidin beads. See methods for details concerning the experiments and analyses. e ) Summary of the gene ontologies (GOs; cellular components) for the proteins biotinylated upon live uptake of NbLumSyt1-APEX2 (for a detailed list, see Supplementary Table 1). As expected, synaptic components and membrane GO terms were overrepresented. f ) Possible interactors identified via live-cell proteomic mapping and enrichment vs. input and vs. IP control. Cntfr was found to be the most enriched candidate, together with other proteins that could be studied in future works. g ) Super-resolution stimulation emission depletion (STED) imaging reveals that ~ 20% of boutons labeled with live uptake are also positive for Cntfr. In this case, for cross-validation purposes, live uptake was performed with the 604.2 Syt1-luminal antibody. h ) Proximity ligation assay (in situ PLA) using antibodies against the luminal portion of Syt1 and anti-Cntfr confirmed the close proximity of these two proteins. A primary antibody against a protein not expressed in hippocampal neurons (Ribeye) was used as the negative control. i ) Blocking the network activity of primary hippocampal neurons with tetrodotoxin (TTX) for 2 h decreases the in situ PLA signal between Syt1 and Cntfr. Stimulation with the ligand of Cntfr (Cntf; 8 nM) for 2 h does not change the PLA signal between Syt1 and Cntfr. j , k ) Stimulation of neurons with Cntf for 24 h increases SV exo-endocytosis. Scale bars: 10 μm in a ; 500 nm in c ; 5 μm in g-j . The error bars indicate the means ± SEMs for panel d, and the 5th or 95th percentile for box plots; ** p < 0.01; *** p < 0.001

Article Snippet: At 14 days in vitro (DIV), the neurons were incubated either in conditioned medium only (control) or in conditioned medium supplemented with 8 nM ciliary neurotrophic factor (CNTF, C-245 Alomone Labs) for 2–24 h or with 3 μM tetrodotoxin (TTX, 1069 Tocris) for 2 h. As a proxy for neuronal activity, neurons were exposed to an anti-Syt1 luminal primary antibody directly labeled with ATTO647N (1:200, 105 311AT1 clone 604.2 Synaptic Systems) in conditioned medium for 30 min at 37 °C.

Techniques: Western Blot, Labeling, In Situ, Electron Microscopy, Control, Membrane, Imaging, Biomarker Discovery, Proximity Ligation Assay, Negative Control, Blocking Assay, Activity Assay