Hyperfilm mp

Proteins separated on the SDS–PAGE gel under reducing conditions were transferred onto an Immobilon‐P PVDF membrane (Merck Millipore, Bedford, MS) by wet transfer blotting methods. The membrane was incubated in 10 mM phosphate buffered saline (PBS) (pH 7.5) containing 0.1% Tween 20 and 5% skim milk for blocking. For immunoblotting using rabbit anti‐Pru p 7 peptide antibodies, antibodies were diluted 1:1000 in the same blocking buffer as for the primary antibody. Then, horseradish peroxidase‐labeled goat anti‐rabbit IgG (KPL; SeraCare, Milford, MA) was used as a secondary antibody. IgE‐immunoblotting used patient sera (1:10 dilution) in the same blocking buffer as the primary antibody. Then, horseradish peroxidase‐labeled goat anti‐human IgE (KPL; SeraCare) was used as a second antibody. Detection of the final signal was performed using an ECL Western Blotting Detection kit (GE Healthcare, Little Chalfont, UK), following the manufacturer's instructions, and recorded onto X ray films (Hyperfilm MP, GE Healthcare).

Cells were treated with CP and, after different times, were collected, centrifuged and washed with ice cold phosphate-buffered saline (PBS). The pellet was then resuspended in lysis buffer as described [47 ]. The protein concentration in the supernatant was determined using the BCA protein assay (Pierce, Italy). Equal amounts of protein (10 μg) were resolved using SDS-PAGE gel electrophoresis (Criterion precast Tris-HCl gel, BioRad, Italy) and transferred to PVDF Hybond-p membranes (GE Healthcare, Italy). Membranes were blocked with 2% ECL-Blocking Solution (GE Healthcare, Italy) for 2 hours at room temperature. Membranes were then incubated with primary antibodies against Bcl-2, PARP, procaspase-9, cleaved caspase-7, GRP78, (Cell Signaling, Italy), cleaved caspase-12 (Abcam, UK), β-actin (Sigma Aldrich, Italy), and cleaved caspase-3 (Novus Biologicals, Italy) overnight. Membranes were then incubated with peroxidase-conjugated secondary antibodies (Invitrogen, Italy) for 60 min. All membranes were visualized using ECL Select (GE Healthcare, Italy) and exposed to Hyperfilm MP (GE Healthcare, Italy). To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

RNA or DNA transfected cells were lysed at 48 h and 32–40 h post-transfection, respectively, in NuPAGE LDS sample buffer (Invitrogen) containing 0.71 M 2-mercaptoethanol and heated for 10 min at 95°C. Proteins were loaded onto NuPAGE 10% or 12% Bis-Tris gels (Invitrogen), separated by SDS-PAGE in MOPS SDS running buffer (Invitrogen) and transferred to PVDF or nitrocellulose membranes. Membranes were saturated in DPBS (Invitrogen) containing 0.1% Tween-20 (PBS-T) and 5% dry skimmed milk at RT for 1 h, prior to incubation for 1 h at RT or overnight at 4°C with one of the following monoclonal antibodies or antisera diluted in PBS-T containing 1% dry skimmed milk: anti-NS2^JFH1 (1:2000), anti-V5 (1:4000), anti-ST (0.05 μg/ml), anti-GFP (0.125 μg/ml), or anti-HAep (0.25 μg/ml). Following incubation for 1 h at RT with peroxidase-conjugated (chemiluminescence, GE Healthcare) or Dylight 680 or 800nm-conjugated (fluorescence, Thermo Scientific) anti-mouse or anti-rabbit antibodies, proteins were visualized using ECL Prime Western Blotting Detection Reagent followed by exposure to Hyperfilm MP (GE Healthcare) or an infrared scanner imager (Odyssey CLx, Li-Cor), respectively. Quantifications were performed on images acquired with the latter system using Image Studio Lite software.

LMMP were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 μM NaVO4, 1 mM NaF) containing a mixture of protease inhibitors (0.5 mM EDTA, 0.1 mM PMSF, 1 μM leupeptin, 150 nM aprotinin) (Brun et al., 2013 (link)). Samples were incubated 30 min at 4°C. Particulate material was removed by centrifugation (15,000 × g, 5 min at 4°C) and protein concentration was determined in the supernatants using the bicinchoninic acid assay (Thermo Scientific, MA, USA). Proteins (40 μg/line) were fractionated through SDS-PAGE gel and immobilized onto PVDF membrane (Bio-Rad Laboratories). Unspecific bindings were blocked for 1 h in 5% non-fat dry milk dissolved in PBS and added with 0.05% Tween20. PVDF membranes were then probed with specific antibodies (Table 1). Immune-complexes were revealed using horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich, Italy; Table 1) and enhanced chemiluminescent substrate (ECL, Millipore, Italy). Images were captured using Hyper Film MP (GE Healthcare, Italy). Antibody against mouse β-actin (Sigma) was used as loading control. Densitometric analysis was performed using the ImageJ software (US National Institutes of Health).

Forty-eight hours after transfection, cells were harvested with ice-cold lysis buffer (0.2 M KCl, 0.03 M Tris, pH 7.25) containing proteinase inhibitor cocktail (Roche Diagnostics). Lysates were boiled in equal volumes of loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% ß-mercaptoethanol), and 20 μg of protein was separated in a 8–12% gradient Tris-glycine gradient gel (Novex, San Diego, CA, USA) under reducing conditions and transferred to nitrocellulose membranes (GE Healthcare, Piscataway, USA). Afterwards, membranes were blocked at room temperature with PBS containing 0.1% Tween and 5% non-fat dry milk to block non-specific binding for 2 h. Membranes were incubated with primary antibodies (rabbit anti-human ß-actin, Cell Signaling Technology, Danvers, USA; mouse anti-human UHRF1, generously provided by Dr. C. Bronner, Institute of Genetics and Molecular and Cellular Biology, Strasbourg, France) at 4 °C overnight, followed by a 1-h incubation with the corresponding secondary antibodies (goat anti-rabbit-HRP and goat anti-mouse-HRP, both Dako Cytomation, Hamburg, Germany) at room temperature. For chemiluminescent detection, the ECL Plus Western detection kit (GE Healthcare) was used. Protein bands were detected by autoradiography using the high-performance autoradiography Hyperfilm™ MP (GE Healthcare).