Mp imaging system

The protein concentration of different cell fractions was quantified using the Bradford assay. Equal amounts of protein samples were subjected to SDS-PAGE and transferred to nitrocellulose membrane (Amersham Protran-GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). After blocking non-specific binding of antibody with 5% non-fat milk, blots were probed with one of the following antibodies: anti-aIF6 polyclonal rabbit antibodies (1:5,000), anti-aSBDS polyclonal rabbit antibodies (1:10,000), 6×-His Tag Monoclonal Antibody (4E3D10H2/E3; Thermo Fisher Scientific). Primary antibodies were detected by binding horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz Biotechnology), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology), and using an enhanced chemiluminescent visualization system (ECL Western Blotting Substrate, Thermo Fisher Scientific-Pierce Biotechnology, Rockford, IL, United States). 6×-His Tag Monoclonal Antibody and secondary antibodies were diluted according to the manufacturer instructions. The images were captured by a BioRad ChemiDoc. MP Imaging system (Bio-Rad, Hercules, California, United States).

Transfected NSCs or cultured fibroblasts were lysed in RIPA buffer in the presence of protease and phosphatase inhibitors. Protein concentrations were estimated using the Pierce BCA Protein Assay Kit. Equal amounts (20 μg) of proteins were separated on 4–20% Bis-Tris sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) to analyze the C-terminal fragment of APP or full-length APP, respectively. For protein blotting, polyvinylidene difluoride membranes were used after activation in methanol solution. Membranes were incubated overnight with primary antibody diluted in 5% bovine serum albumin. Protein band visualization was performed using a Chemidoc MP imaging system with Image Lab software (Biorad, Copenhagen, DK).
For pTyr IP, total lysates were incubated overnight with mouse pTyr antibody (magnetic bead conjugate). The IP samples were analyzed by WB using rabbit anti-APP, rabbit anti-pan Fyn and rabbit anti-Src pTyr416 (also recognizing Fyn Tyr420).

Variation in 70 bp kDNA-PCR amplification band intensity was assessed by 2.5% agarose gel electrophoresis with GelRed Nucleic Acid gel stain in 1X Tris–Acetate-EDTA buffer (TAE buffer). Invitrogen 100 bp DNA Ladder was used for identification of size amplification band. The amplification bands from different sampling methods were compared with the intensity of the positive control band using Leishmania (Viannia) braziliensis LTB300 strain in a ChemiDoc MP Imaging System with Image Lab software v5.1 (BIO-RAD).

Supernatants from S. suis cultures were collected after 6 h of growth curves (4.5.) by centrifugation (2100× g, 5 min at room temperature) and stored at −20 °C until usage.
DNase activity of S. suis serotypes was tested in 50 µL supernatant mixed with 50 µL DNase buffer (pH 7.4, 3 mM CaCl₂, 3 mM MgCl₂, 300 mM TRIS) to a final reaction volume of 100 µL. In this sample, 0.5 µg calf thymus DNA (Sigma Aldrich) was incubated. The negative control was THB medium; as positive control, micrococcal nuclease from Staphylococcus aureus (Sigma Aldrich) was used.
Samples were incubated for 6 h at 37 °C. Visual examination of DNase activity was conducted in all assays after incubation with 1% agarose gel electrophoresis (100 V, 30 min) and staining of DNA with Roti- GelStain (Roth GmbH & Ko. KG, Karlsruhe, Germany). The gels were analyzed with a Bio-Rad MP Imaging System.