Linear diagram of prion protein showing epitope location of PrP-specific monoclonal antibodies used on N2a/22L cells and in PMCA.

<p>Linear diagram of prion protein showing epitope location of PrP-specific monoclonal antibodies used on N2a/22L cells and in PMCA.</p

Characterization of Mabs.

<p>Characterization of Mabs.</p

Synergy of APAC and SLK on ex vivo platelet aggregate dissolution and platelet-rich clot lysis.

<p>(A) Platelet aggregates were prepared as described above. Black bars refer to platelet aggregate size at zero time. The 3 companion hatched bars refer to platelet aggregate size at 2 hours. Concentration of SLK and APAC was at 0.025 µM. SLK+APAC double hatched bar refers to final SLK and APAC concentration at 0.025 µM each. (B) Platelet-rich clots were formed on the wells of ELISA plate. The clots were treated with 0.025 µM Ctl scFv (13CG2) or SLK or APAC or SLK+APAC. SLK+APAC refer to final SLK and APAC concentration at 0.025 µM each. The relative clot turbidity was calculated by detecting the decrease of the absorbance at OD405. The mean±SD. came from 3 separate experiments. Each time point represents 5 measurements.</p

Generation of a bifunctional APAC capable of homing to active platelets.

<p>(A) Schematic diagram describing cloning strategy for the fusion construct. (B) Plasmid Construct. (C) 12% SDS-PAGE analysis of five aliquots of purified fusion protein (∼60 KD) with Ni-column following induction by 1 mM IPTG.</p

A. Mab Inhibition of PrP^Sc in N2a/22L Cells.

<p>N2a/22L cells were incubated with purified Mabs for 96 hrs. Cells were harvested and PK-treated lysates were western blotted (<b>see </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041626#pone-0041626-g002" target="_blank"><b>Figure 2B</b></a><b>for representative western blots</b>). PrP^Sc western blots were quantitated and the amount of inhibition was determined relative to N2a/22L control cultures. The controls consisted of cells both in the absence of Mab and in the presence of normal mouse IgG. The % PrP^Sc inhibition plotted represents the mean ± SD from three independent experiments as described in Methods.</p

A. Western blot of 263K brain homogenate that was used as the seed for PMCA.

<p>Dilutions of the brain homogenate was prepared and either untreated (lanes 1 and 2) or PK-treated (lanes 3 and 4) prior to SDS-PAGE and western blotting. A 10−3 dilution prior to and after PK demonstrates the three protein banding pattern typical for 263K brain homogenate whereas no bands are visible at a 10−4 dilution of the same homogenate. <b>B.</b> Western blotting of the PMCA products following sPMCA40 in the absence and presence of PrP-Specific Mabs. Fourty cycles of serial PMCA was carried out in the absence or presence of Mabs as described in the text. Each Mab was added at a final concentration of 12, 25, and 50 µg/ml. Following PK treatment, the PMCA products were subjected to SDS-PAGE, western blotted and immunostained for PrPSc. The protein bands were quantitated and the level of PrPSc inhibition, relative to the no Mab and normal mouse IgG controls, were determined.</p

Influence of Mabs 3F4 and 5D6 on PrP^Sc formation following sPMCA₄₀.

<p>Mabs 3F4 and 5D6 were added to sPMCA₄₀ at final concentrations of 0–50 µg/ml. The PMCA products were PK treated and western blotted. The PrP^Sc was quantitated and the level of PrP^Sc inhibition was determined relative to control reactions.</p

Characterization of a bifunctional APAC agent capable of homing to active platelets.

<p>(A) Binding assay of APAC to platelets. (a) resting mouse platelets; (b) ADP-stimulated mouse platelets; (c) resting human platelets; (d) ADP-stimulated human platelets, respectively. (B) Binding assay of APAC versus A11 to activated human platelets determined by flow cytometry. Data were presented as mean ±SD (n = 3). (C) Effect of APAC on platelet fragmentation. Bars labeled 2, 3 and 4 after 1 refer to serial doubling dilutions of 1∶2–1∶16 respectively (0.1 µM APAC), n = 4, SD is given. (D) Dissolution of ex vivo collagen-induced platelet aggregates with APAC. Data and SD are given for 3 separate experiments at 0.1 µM reagent in which each time point represents 5 measurements.</p

Additional file 1 of High-fat diet-induced atherosclerosis promotes neurodegeneration in the triple transgenic (3 × Tg) mouse model of Alzheimer’s disease associated with chronic platelet activation

Additional file 1: Table S1. Antibodies used in this study. TableS2. Serum differentially expressed proteins between high-fat diet-treated (H) and normal-chow-treated (N) 3 × Tg mice. Figure S1. Gene ontology (GO) term and pathway analyses of significantly changed proteins by Metascape revealed enrichment of proteins of several pathways related to complement and coagulation cascades, blood coagulation, platelet degranulation, and cell-substrate adhesion. Figure S2. Initial CAA lesions in HFD-treated 3 × Tg mice. Figure S3. Safety of A11 injection on other organs.

MOESM1 of Secernin-1 is a novel phosphorylated tau binding protein that accumulates in Alzheimer’s disease and not in other tauopathies

Additional file 1: Figure S1. Confirmation of α-SCRN1antibody specificity. a Western Blot analyses of fresh frozen frontal cortex tissue from n = 2 AD and n = 2 cognitively normal samples with PHF1 (pTau ser396/ser404) and two different α-SCRN1 antibodies specifically labelling the 46KDa full-length SCRN1. Immunoblot showed one specific band for SCRN1 and similar SCRN1 levels in AD and cognitively normal samples. Fifteen micrograms protein per sample from total homogenate were loaded. GAPDH was used as loading control. b Absorption assay showing the lack of SCRN1 staining after pre-absorption with human recombinant SCRN1 protein. -ve: negative control (no primary antibody)