Tgx stain free precast gradient gels

Equal volumes (15 µL) of protein lysates were separated on TGX Stain-Free precast gradient gels (4–20% Mini-PROTEANR TGX Stain-Free Protein Gels, Biorad, Munich, Germany) in Laemmli buffer. Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Biorad) and subsequently membranes were blocked for 2 h in 5% BSA in Tris buffered saline with 0.05% Tween 20 (TBST). Membranes were probed overnight at 4 °C in primary antibodies: STAT3 (124H6) mouse antibody; pSTAT3 (Tyr705) (D3A7) XPR rabbit antibody; MYC antibody; pMYC (Thr58) (E4Z2K) rabbit antibody; beta-actin (8H10D10) mouse antibody (all 1:1000; Cell Signaling Technology, Danvers, MA, USA) in blocking solution. After washing membranes in TBST (3 × 15 min), membranes were probed with appropriate peroxidase-conjugated secondary antibodies (mouse anti-rabbit IgG (D4W3E) antibody and rabbit-anti-mouse IgG (D3V2A) antibody (both Cell Signaling Technology) in TBST for 1 h at room temperature. After final washing (3 × 15 min), blots were developed using a Chemidoc MP Imaging System and the Clarity Western ECL substrate (Biorad). Quantification of protein expression was carried out with ImageLab Software (Biorad) and normalized to the expression of beta-actin.

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)].

Cytosolic liver extracts were prepared by homogenizing fresh tissue in 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, 0.1% Nonidet-P40, protease inhibitor cocktail and PhosSTOP^TM (all Sigma-Aldrich), leaving it on ice for 30 min with occasional vortexing before centrifuging at 4000×g and 4 °C for 5 min. Supernatant protein concentrations were determined with the BCA assay (Thermo Fisher Scientific, Schwerte, Germany). The samples were mixed with loading buffer, denatured at 95 °C for 5 min and separated on TGX Stain-Free Precast gradient gels (Biorad, Munich, Germany) and blotted onto a PVDF membrane. The membrane was blocked with 5% skim milk dissolved in TBS with 0.05% Tween-20 and probed with a primary antibody overnight (MUP antibody sc-21856, Santa Cruz Biotechnology Inc., Heidelberg, Germany; p-AMPK antibody CS-2523, Cell Signalling, Leiden, The Netherlands; Actin antibody ab3280, Abcam, Cambridge, UK) followed by a secondary antibody (anti-goat sc-2354: Santa Cruz Biotechnology Inc., Heidelberg, Germany, anti-mouse 170–5047; anti-rabbit 170–5046: Biorad). The bands were visualized with ECL reagent (Thermo Fisher Scientific) in a ChemiDoc XRS system (BioRad).