PMC

Clotting was monitored as the change in absorbance at 490 nm over time during incubation at 37°C. Measurements were performed every 6 s, and no curves have been fitted. Some results are given in several panels for comparison. Shown are the mean and 95% CI for six replicates in two blocks with different donors of saliva and plasma for blocks. (a) Pathways of coagulation. Plasma was diluted to a final concentration of 10% in saliva (S [black]), saliva with an inhibiting antibody against FVII (S, aFVII [red]), saliva with a control antibody (S, ab [green]), saliva with an inhibitor of FXII/PK (S, iFXII [blue]), or plasma that had been passed through a 3-kDa filter (P<3 [yellow]). Saliva with no plasma added was used as a negative control (−P [brown]). (b) Fractions of saliva. Plasma was diluted to a final concentration of 10% in saliva (S [black]), saliva passed through a 3-kDa filter (S<3 [blue]), or the fraction of saliva recovered with a 3-kDa filter (i.e., the protein fraction) that had been washed and diluted to the original concentration in an NaCl-CaCl₂ solution with the same ionic strength as plasma, and the same calcium concentration as protein-depleted saliva and plasma (S>3 [red]). (c) Protein fraction. Plasma was diluted to a final concentration of 10% in the fraction of saliva recovered with a 3-kDa filter (i.e., the protein fraction) that had been washed and diluted to the original concentration in an NaCl-CaCl₂ solution with the same ionic strength as plasma and the same calcium concentration as protein-depleted saliva and plasma. To this mixture, the following was added: nothing (S>3 [black]), control antibody (S>3, ab [green]), anti-FVII antibody (S>3, aFVII [red]), or inhibitor of FXII/PK (S>3, iFXII [blue]). (d) Non-protein fraction. Plasma was diluted to a final concentration of 10% in saliva passed through a 3-kDa filter. To this mixture, the following was added: nothing (S<3 [black]), anti-FVII antibody (S<3, aFVII [red]), or an inhibitor of FXII/PK (S<3, iFXII [blue]). (e) Ionic strength. Plasma was diluted to a final concentration of 10% in saliva passed through a 3-kDa filter (S<3 [black]), plasma passed through a 3-kDa filter (P<3 [blue]), plasma passed through a 3-kDa filter and diluted to an ionic strength similar to that of saliva while holding the calcium concentration constant (P<3, diluted [red]), or saliva passed through a 3-kDa filter with NaCl added to a final ionic strength similar to that of plasma (S<3, salt [yellow]). (f) Combining components. Plasma was diluted to a final concentration of 10% in saliva (S[black]), an NaCl-CaCl₂ solution with the same ionic strength as plasma and the same calcium concentration as protein-depleted saliva and plasma (Hsalt [blue]), an NaCl-CaCl₂ solution with the same ionic strength as saliva and the same calcium concentration as protein-depleted saliva and plasma (Lsalt [green]), the fraction of saliva recovered with a 3-kDa filter that had been washed and diluted to the original concentration in an NaCl-CaCl₂ solution with the same ionic strength as plasma and the same calcium concentration as protein-depleted saliva and plasma (S>3, Hsalt [yellow]), or the fraction of saliva recovered with a 3-kDa filter that had been washed and diluted to the original concentration in an NaCl-CaCl₂ solution with the same ionic strength as saliva and the same calcium concentration as protein-depleted saliva and plasma (S>3, Lsalt [red]).

Immunoprecipitation of Gγ3 and Gγ3ΔCys candidates. (A) Immunoprecipitation of the Gγ3 candidate from solubilized plasma membrane proteins of the wild-type (WT) flower using an anti-Gγ3 domain antibody. Molecular weight marker (lane 1); 10 μg of protein of the plasma membrane fraction of the WT (lane 2); the immunoprecipitation product of solubilized plasma membrane proteins and anti-Gγ3 domain antibody (lane 3); control experiment (buffer in place of the membrane protein; lane 4). (B) Immunoprecipitation of the Gγ3ΔCys candidate from the solubilized plasma membrane proteins of the Minute (Mi) flower using an anti-Gγ3 domain antibody. Molecular weight marker (lane 1); 10 μg of protein of the plasma membrane fraction of Mi (lane 2); the immunoprecipitation product of the solubilized plasma membrane proteins of Mi and the anti-Gγ3 domain antibody (lane 3); control experiment (buffer in place of the membrane protein; lane 4).

ZmVPP5 is found in the plasma membrane, vacuolar membrane, and nuclei (A) Detection of ZmVPP5 in the plasma membrane. H⁺‐ATPase was used as a plasma membrane localization marker, and BIP was used as an ER marker. 1, total protein; 2, supernatant; 3, endomembrane fraction; 4, plasma membrane fraction. (B) Detection of ZmVPP5 in the vacuolar membrane fraction and nuclei fraction. Histone 3 was used as a nuclear marker, and V‐ATPase was used as a vacuolar membrane marker. 1, total protein; 2, vacuolar membrane fraction; 3, nuclei fraction.

Nanoparticle tracking analysis (NTA) of the number of small EVs (< 200 nm) in each fraction isolated from the plasma of (a) humans and (b) felines by SEC. Protein concentration (-0-) of each fraction isolated by SEC from the plasma of (a) humans and (b) felines by BCA. (a) The enrichment of small EVs in human plasma within fractions 7–10 is shown. The protein concentration also increases in later fractions, with fractions 11 and 12 containing substantially more protein than previous fractions. (b) The enrichment of small EVs in feline plasma within fractions 7–10 is shown. The protein concentration increases in later fractions, with fractions 11 and 12 containing substantially more protein than previous fractions. This figure confirms that the majority of small EVs are contained in fractions 7–10, while fractions 11 and 12 from both human and feline plasma contain substantially more co-isolated contaminating protein than previous fractions. A high protein to particle ratio has been shown to indicate contamination by plasma proteins.