Fusion fx7

The APAP-treated cells were lysed in RIPA with protease and phosphatase inhibitors for 30 min. The cells were centrifuged at 9000× g for 10 min at 4 °C and the supernatant was collected. The total protein content of the supernatant was determined using a BCA kit. After the BCA assay, the supernatant was fixed in 5 × loading buffer in boiling water for 10 min. The proteins were separated using SDS-PAGE gels and then transferred onto PVDF membranes. After 1 h of blocking with 5% nonfat milk, the membranes were incubated with appropriate primary antibodies overnight at 4 °C. Subsequently, the membranes were washed two times with TBST and hybridized with horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulin IgG secondary antibodies for 2 h [43 (link)]. After washing with TBST three times, the protein bands were visualized by an imaging system (VILBER Fusion FX7, Vilber Lourmat, Marne-la-Vallee, France) using enhanced chemiluminescence detection reagents. The band intensities were quantified using Image J gel analysis software.

The cells were lysed on ice with WB/IP lysis buffer containing a protease inhibitor cocktail (Roche, Cat. No. 04693116001). Protein samples were normalized to equal amounts of GAPDH/ACTB, separated by SDS-PAGE, and transferred onto 0.22 µm polyvinylidene fluoride membranes (Merck, Cat. No. ISEQ00010), which were blocked in 5% skimmed milk at RT for 2 h. The membranes were incubated with the primary antibodies at 4°C overnight and then incubated with the corresponding secondary antibodies (conjugated with HRP) at RT for 1 h. After washing, IB results were visualized with enhanced chemiluminescence reagents (NCM Biotechnology, Cat. No. P10300) and imaged using a chemiluminescence imaging system (Fusion FX7; VILBER, Paris, France). Representative images were shown.

After amplification, protease-resistant prion protein was detected by western blot as described previously^{61 (link)}. After proteinase K digestion (200 µg/mL) for 60 min at 45 °C and denaturation at 100 °C in SDS–PAGE denaturing buffer, samples were run on 12% polyacrylamide gel electrophoresis, before being electro-transferred onto PVDF membrane. Western blot (using the SNAP ID system, Millipore) was performed using 3F4 (mAb 3F4, epitope 109–112 of human PrP—Ozyme, France), 12B2 (mAb 12B2, epitope amino acid residues 89–93 of human PrP—Wageningen Bioveterinary, Netherlands), 9A2 (mAb 9A2, epitope amino acid residues 99–101—Wageningen Bioveterinary, Netherlands) or 6D11 (mAb 6D11, epitope 93–109 of human PrP sequence—Ozyme, France), and anti-mouse IgG peroxidase-linked secondary antibody (GE Healthcare, UK) linked to a chemiluminescent reaction (ECL blotting detection reagent, GE Healthcare, France), and imaged using films except for Fig. 3c using the imaging system Fusion FX7 (Vilber, France). The detection limit was determined visually after a maximum time exposure of 30 min and as result the dilution scored positive when the three characteristic PrP^res bands were observed.

Cells were extracted with ice-cold Nonidet P-40 lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 30 mM MgCl₂, 1% Nonidet P-40, 1 mM DTT) and a mixture of protease and phosphatase inhibitors. Lysates were then centrifuged for 10 min at 14,000 g at 4°C. 30 µg of protein from each supernatant was subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane which was blocked with 3% BSA for 1 h at room temperature. Immunoblotting was performed with primary specific antibodies listed in S1 Table. The peroxidase-conjugated secondary antibody was incubated for 1 h at room temperature and developed with enhanced chemiluminescence using Fusion FX7 (Vilber, Germany).

NB cells and tissues were processed for Western blotting as described [48 (link)]. In brief, cell lysates of NB tissues and control tissues were blotted on a nitrocellulose membrane and PlGF-1 and -2 protein expression were detected with a rabbit polyclonal antibody against PlGF (Abcam, Cambridge, UK) and VEGF-A protein expression was detected with a rabbit polyclonal antibody against VEGF-A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), before incubation with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, Buckinghamshire, UK). To measure the secretion of (s)VEGFR2 in SK-N-AS cells following infection compared to RFP controls, supernatants from Ad(s)VEGFR2 and AdRFP infected HEK293 cells were loaded on 7.5% acrylamide gels and semi-dry-blotted on nitrocellulose membranes and (s)VEGFR2 was detected with goat anti-mouse VEGFR2/FLK-1 antibody (LifeSpan BioSciences, Seattle, WA, USA) followed by a donkey anti-goat HRP conjugated IgG secondary antibody (LifeSpan BioSciences). Proteins were immunodetected by chemiluminescence (Ace Glow, Peqlab, Erlangen, Germany), scanned using FUSION-FX7 (Vilber Lourmat, Marne-la-Vallée, France) and quantified by Fusion-CAPT-Software 16.07 (Vilber Lourmat).