Prolastin

Prolastin (Grifols, Barcelona, Spain, for more information see Prolastin-C.pdf">http://www.grifols-pi.info/inserts/Prolastin-C.pdf) was used as a source for A1AT. Prolastin (1 g) was solubilized with 40 ml ultrapure water, aliquoted and stored at -80°C until use.
To obtain Aβ oligomer-enriched preparations, 1 mg Aβ_1-42 (Bachem, Bubendorf, Switzerland) was dissolved in 10% ammonia, aliquoted, freeze dried and stored at -80°C. Before use, Aβ_1-42 aliquots were dissolved in sterile water (1 mg/ml), and then 100 mM Tris and 50 mM NaCl were added to obtain a 58 μM solution. A magnetic stirrer was added and the Aβ solution was stirred for 48 hours at 1,400 rpm at room temperature.
Antibodies against the following proteins were used: phosphorylated (phospho)-p38, phospho-p44/42, phospho-JNK (c-Jun N-terminal kinase)/SAPK (stress-activated protein kinase), phospho-CREB (all Cell Signaling, Danvers, MA, USA), Aβ (clone 6E10, Covance, Princeton, NJ, USA), A1AT, Vinculin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Novus Biologicals, Littleton, CO, USA) and horseradish peroxidase-conjugated secondary antibody goat anti-mouse, goat anti-rabbit (both Cell Signaling, Danvers, MA, USA) and donkey anti-goat (Santa Cruz Biotechnology, Dallas, TX, USA).

Purified human plasma pooled A1AT (Prolastin, Grifols) was used for in vitro experiments. To remove high molecular mass proteins, specifically IgA [16 (link),23 (link),24 (link)] Prolastin was first diluted in phosphate-buffered saline (PBS) and repurified by ultrafiltration using a centricon-100 kDa and centricon-30 kDa cutoff. The IgA-free preparation of A1AT was subjected to quantitative analysis using bicinchoninic acid assay (BCA assay, Pierce BCA Protein Assay Kit, Thermo Scientific) and to qualitative evaluation using Western blot analysis with specific anti-A1AT and anti-IgA antibody.