Liver Protein Quantification via Western Blot

Liver protein lysates were loaded on TGX Stain-Free Precast gradient gels (Biorad, Munich, Germany) (40 µg protein per lane) and separated by SDS precast polyacrylamide gel electrophoresis, as described in detail in previous publications [55 (link), 63 (link)]. Following the protein transfer onto a polyvinylidene difluoride membrane with the Trans Blot Turbo™ System (Biorad), the target proteins were identified using the following primary monoclonal antibodies (all purchased from Merck): anti-phospho-cytokeratin-8 (Ser73) (clone LJ4), anti-cytokeratin 8 (clone TROMA-1) and anti-HMGB1 (clone 3K6). Secondary horseradish peroxidase-conjugated antibodies were purchased from Biorad (Immun-star goat anti-mouse IgG (1705047) and anti-rabbit IgG (1705046)) and Merck (anti-rat IgG (A9037)). Target bands were visualized by chemiluminescent ECL Western blot substrates (Fisher Scientific, Schwerte, Germany) in a ChemiDoc XRS system (Biorad) and band intensity was analyzed using the Image Lab 4.1 software (Biorad). Target proteins were normalized by the total protein load per lane, measured as membrane fluorescence and as described previously [55 (link)]. The determination of hepatic APOE has been performed in a previously published study and the values are according to Rueter et al. [57 (link)].