Mouse monoclonal anti αs 211 antibodies

To probe αS conformers, 0.7 μg of each sample (2 μL) was spotted onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked for 30 min with 1.0% bovine serum albumin in TBS/TWEEN 0.1% and then incubated overnight at 4 °C with rabbit polyclonal anti-oligomer A11 antibodies (1:1000, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-amyloid fibrils OC antibodies (1:1000, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-aggregated αS 5G4 antibodies (1:1800, Sigma-Aldrich), or with mouse monoclonal anti-αS 211 antibodies (1:1800, Santa Cruz Biotechnology, Dallas, TX, USA). The immunolabelled blots were finally incubated with the proper secondary antibodies conjugated with horseradish peroxidase (Abcam, Cambridge, UK) and then detected by using a Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and ImageQuant™ TL software (GE Healthcare, Chicago, IL, USA).

SH-SY5Y cells were treated with SF and OB*at 0.3 µM for 24 h. Membrane and cytoplasmic fractions were obtained as previously described with minor modifications [21 (link)]. Briefly, the cells were homogenized in PBS containing 9.0% sucrose with three freeze–thaw cycles, 5.0 s sonication on ice and centrifugation at 700× g for 10 min at 4 °C. The membrane fraction was pelleted by supernatant centrifugation at 110,000× g for 1 h at 4 °C and separated from the cytoplasmic one. The protein content in the fractions was measured by the method of Bradford [22 (link)]. Thirty µg of each fraction were run on 4–20% Mini-PROTEAN TGX precast gels (Bio-Rad, Hercules, CA, USA) and the separated proteins were blotted onto a Supported Nitrocellulose Membrane (Bio-Rad). The blotted membranes were then blocked in 1.0% (w/v) BSA in TBS-Tween (0.1% Tween 20 in 20 mM Tris–HCl buffer, pH 7.5, containing 100 mM NaCl) and then incubated with mouse monoclonal anti-αS 211 antibodies (1:250, Santa Cruz Biotechnology). After extensive washing, the membranes were incubated with peroxidase-conjugated anti-mouse secondary antibodies (Abcam) for 1 h and the immunolabelled bands were detected using a Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) and ImageQuant™ TL software (GE Healthcare).