Journal: Medicina

Article Title: Bacterial Production of Recombinant Coagulation Factor VIII Domains

doi: 10.3390/medicina59040694

Figure Lengend Snippet: ( A ) Agarose gel electrophoresis analysis of DNA products after amplifying insert DNA (rF8-A2-or rF8-A3-encoding gene) and vector DNA (linearized pGEX-4T1); ( B ) SDS-PAGE analysis of rF8-A2 or rF8-A3. The black arrow indicates the target protein; ( C ) Schematic representation of the ELISA; ( D ) ELISA signal obtained from rF8-A2 with rabbit anti-F8 polyclonal antibody; ( E ) ELISA signal obtained from rF8-A2 with mouse anti-A2 monoclonal antibody; ( F ) ELISA signal obtained from rF8-A3 with rabbit anti-F8 polyclonal antibody. Error bars represent ±1 SD ( n = 3). SD = standard deviation. GST, HRP, IgG, ABS, pAb, and mAb indicates glutathione S-transferase, horseradish peroxidase, immunoglobulin G, absorbance, polyclonal antibody, and monoclonal antibody, respectively.

Article Snippet: After washing using PBS0.05T 3 times, 2000-times diluted rabbit anti-F8 polyclonal antibody (Sino Biological, Beijing, China) or 2000-times diluted mouse anti-F8-A2 monoclonal antibody (Novus Biologicals, Englewood, CO, USA) in PBS was added as a primary antibody and incubated for 1 h at RT.

Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, SDS Page, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Anticoagulant Effect of PGI2S and tPA in Transgenic Umbilical Vein Endothelial Cells Is Linked to Up-Regulation of PKA and PKC

doi: 10.3390/ijms15022826

Figure Lengend Snippet: Expression of anticoagulation factors by HUVEC-mock, HUVEC- PGI2S , and HUVEC- PGI2S-tPA cells, ELISA were performed to measure the levels of ATIII , PLG and Factor VIII in cell homogenates, p values were calculated using one-way ANOVA.

Article Snippet: Enzyme-linked immunosorbent assays (ELISAs) were performed on the cell homogenates using the Human Coagulation Factor VIII (FVIII) ELISA kit (Cusabio, Wuhan, China), Human Antithrombin III (ATIII) ELISA kit (Cusabio) and ELISA kit for plasminogen (USCN, Wuhan, China) following the manufacturer’s protocol.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: TRIM28 regulates the coagulation cascade inhibited by p72 of African swine fever virus

doi: 10.1186/s13567-024-01407-6

Figure Lengend Snippet: ASFV infection regulated coagulation and related factors in vivo . A Evolution of rectal temperature, B clinical score in animals (6 animals) IM infected with 10 2.5 HAD 50 of ASFV HLJ/18. C viremia, viremia values are expressed as copies/mL, sensitivity of virus detection: > 1000 copies/mL, and D mortality. Dpi: days post-infection. E Sketch of the coagulation activation process. F ASFV-positive serum samples from each pig at the indicated time points were collected for detection of F10 and G F10 activity. H ASFV-positive serum samples from each pig at the indicated time points were collected for detection of F2 and I D2D by ELISA. Statistical data were analysed by t test analysis of variance (* p < 0.05, ** p < 0.01, and *** p < 0.001). All the data are expressed as the mean ± SD.

Article Snippet: The pig coagulation F10 ELISA kit, pig coagulation factor II ELISA kit, and D-dimer ELISA kit were purchased from Cusabio Company (Wuhan, China).

Techniques: Infection, Coagulation, In Vivo, Virus, Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: TRIM28 regulates the coagulation cascade inhibited by p72 of African swine fever virus

doi: 10.1186/s13567-024-01407-6

Figure Lengend Snippet: ASFV-encoded proteins regulated F10 expression and activity . A Several encoded proteins expressed in the baculovirus expression system were added to Huh7 cells at the same concentration for 24 h prior to supernatant and cell lysate collection. The mixtures were detected for F10 activity. B Huh7 cells were transfected with plasmids expressing ASFV-encoded proteins, including pK205R, pE199L, pEP153R, p30, p54, and p72, and then, the cells and supernatants were collected for detection of F10 expression by western blotting, C and secretory F10 expression by ELISA. For all figures, the experiments were repeated at least three times with similar results. The data are presented as the mean ± SD from one single experiment. Statistical significance was determined by Student’s t test (* p < 0.05).

Article Snippet: The pig coagulation F10 ELISA kit, pig coagulation factor II ELISA kit, and D-dimer ELISA kit were purchased from Cusabio Company (Wuhan, China).

Techniques: Expressing, Activity Assay, Concentration Assay, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: TRIM28 regulates the coagulation cascade inhibited by p72 of African swine fever virus

doi: 10.1186/s13567-024-01407-6

Figure Lengend Snippet: Functional domain of p72 . A Schematic of truncated p72 expression. B Confocal microscopy of truncated proteins in Huh7 cells; the target proteins are stained red. DAPI was used to stain the sections blue. Bar, 20 μm. C Truncated plasmids containing amino acids 1–150, 151–422, and 423–646, D 151–450, E and 423–646 of p72 with an HA tag at the C-terminus were transfected (0.5, 1, or 2 μg) into Huh7 cells. At 24 h post-transfection, the cell lysate was collected and subjected to western blot analysis, and the F cell supernatant was collected for detection of secretory F10 expression via ELISA. G Mutant plasmid construction schematic. The sequences of the six p72 mutants ranged from 423–450 aa, and every 10 amino acids were randomly mutated to alanine, which were named 101, 102 (423–432 aa), 201, 202 (433–442 aa), 301, and 302 (443–452 aa). H Confocal microscopy of mutant proteins in Huh7 cells. The target proteins are stained green. DAPI was used to stain the sections blue. Bar, 20 μm. I The mutant plasmids were transfected into Huh7 cells at increasing doses (0.5, 1, and 2 μg) . At 24 h post-transfection, the cells were lysed with lysis buffer, and 15 μg of each sample was subjected to western blotting. The band intensities were normalized to those of GAPDH via ImageJ software. The data are expressed as the mean intensity ratio ± SD of F10 to GAPDH. The experiment was performed in triplicate, and images from three independent experiments were plotted. J The cell supernatant was also collected for detection of secretory F10 expression via ELISA. Statistical data were analysed by t test variance (* p < 0.05, ns represents nonsignificant p > 0.05).

Article Snippet: The pig coagulation F10 ELISA kit, pig coagulation factor II ELISA kit, and D-dimer ELISA kit were purchased from Cusabio Company (Wuhan, China).

Techniques: Functional Assay, Expressing, Confocal Microscopy, Staining, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis, Plasmid Preparation, Lysis, Software

Journal: Nutrients

Article Title: Is the Habitual Dietary Intake of Foods of Plant or Animal Origin Associated with Circulating Hemostatic Factors?—Results of the Population-Based KORA-Fit Study

doi: 10.3390/nu16030432

Figure Lengend Snippet: Overview of measured laboratory variables.

Article Snippet: Factor VIII , Photometry , Coagulation factor VIII deficient plasma, Pathromtin SL, CaCl 2 , Siemens Eschborn , 70–150%.

Techniques: Activity Assay, Immunoturbidimetry Assay, Coagulation, Clinical Proteomics, Nephelometric Assay