Ecl prime chemiluminescence kit

Protein samples were separated on 16.5 % Tris-Tricine Criterion gels (Biorad) and subsequently transferred to 0.2 µm nitrocellulose membranes (GE Healthcare). After a boiling step of 4 min in 1× PBS, membranes were blocked in TNT buffer (Tris–HCl 15 mM pH 8, NaCl 140 mM, 0.05 % Tween) with 5 % non-fat dry milk for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-Aβ_1–16 mAb (6E10, 1/5000, Covance), anti-Aβ_1–40 mAb (9682, 1/500, Cell Signaling), anti-Aβ_1–42 mAb (12F4, 1/500, Covance), anti-Aβ_1–43 mAb (9C4, 1/500, Covance), anti-α-tubulin (11H10, 1/5000, Cell Signaling) and anti-β-actin (1/100,000, Abcam). HRP-conjugated anti-mouse or anti-rabbit antibodies (1/10,000, Invitrogen) were used for 1 h at room temperature and detection was performed using ECL or ECL prime chemiluminescence kits (GE Healthcare) and Hyperfilms (GE Healthcare). Bands were quantified using the ImageJ software (Scion Software) and results are expressed as mean ± sem.

The procedure was based on previous reports [21 (link)] with some modifications. Briefly, 20 female heads per biological replicate were homogenized in 100 µL ice-cold 1× PBS buffer supplemented with Complete mini without EDTA protease inhibitor (Roche) using a disposable pellet mixer and a plastic Eppendorf pestle. Samples were then ultra-centrifuged at 78,000g for 1 h at 4 °C. The supernatant, i.e. the PBS-soluble fraction was collected and protein concentration was measured using the BCA protein assay kit (Pierce). One microlitre per sample (corresponding to 1.5 µg of proteins) was spotted onto a 0.2 µm nitrocellulose membrane (GE Healthcare) and let to dry for 30 min. The membrane was then blocked in TBS-low tween buffer (containing 0.01 % Tween) with 10 % non-fat dry milk for 1 h at room temperature, incubated overnight at 4 °C with the A11 anti-oligomer antibody (1/1000, Invitrogen) and then for 1 h at room temperature with HRP-conjugated anti-rabbit antibody (1/10,000, Invitrogen). Detection was performed using ECL prime chemiluminescence kits (GE Healthcare) and Hyperfilms (GE Healthcare).

Infected HEK293 and AGS cell lines were harvested using cell scrapers, and heated at 95 °C for 5 min in 1× Laemmli buffer. Separation of proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on gels with 6–10% polyacrylamide followed by Western blot analysis using ROTI®PVDF membranes (Carl Roth, Karlsruhe, Germany). The membranes were incubated for 1 h at 20 °C with TBS-T buffer (140 mM NaCl, 25 mM Tris–HCl pH 7.4 and 0.1% Tween-20) including either 3% BSA or 5% skim milk [45 (link)] to block non-specific binding sites. For detection, the following antibodies were used: mouse monoclonal antibody to GAPDH was obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Phosphorylated and total CagA proteins were identified by successive probing of the blots with the mouse monoclonal α-pan-phosphotyrosine antibody PY99 (Santa Cruz Biotechnology) and rabbit polyclonal antibody against CagA (Austral Biologicals, San Ramon, USA) [46 (link)]. Polyvalent horseradish peroxidase (HRP)-coupled secondary goat antibodies were used to detect mouse and rabbit primary antibodies (Thermo Fisher Scientific, Massachusetts, USA). Subsequently, the blots were visualized using the ECL Prime chemiluminescence kit from GE Healthcare as described [47 (link), 48 (link)].