Mini pvdf transfer pack

Cells were collected by centrifugation at 10,000 g for 2 min and were suspended with BugBuster protein extraction reagent (Novagen) for protein extraction according to the manufacturer's instructions. The supernatant of the cell crude extract was subjected to a protein assay (Pierce BCA protein assay reagent, Thermo Fisher Scientific) according to the manufacturer’s protocol. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of cell pellets, which were suspended with phosphate-buffered saline (PBS) buffer (Sigma), was performed with a precast gel (12% Mini-PROTEAN TGX Precast Gel, Bio-Rad) and was blotted with a semidry system (Trans-Blot Turbo Transfer System and mini PVDF Transfer Pack, Bio-Rad). The proteins were detected with primary antibodies (0.1–0.5 μg/mL) derived from mice or rabbits followed by the relevant IgG-HRP conjugates (1,000- to 5,000-fold dilution). After development with a chemiluminescent substrate (Advance Western Blotting Detection Kit, GE Healthcare), the proteins were detected using a luminescent imaging analyzer (Image Quant 350, GE Healthcare). The standard curves for the quantitative evaluation of cellular GFP and S1 ranged from 1–50 ng per lane. The number of cells loaded per lane varied from 1×10⁶ to 2×10⁷ cells according to the preliminary estimated sensitivity in detection.

After 2-DE, gels were either stained with colloidal Coomassie blue R solution (12% trichloroacetic acid, 5% ethanolic solution of 0.035% Serva blue R-250) or transferred onto PVDF membranes (Mini-PVDF transfer pack, Bio-Rad) in the Trans-Blot Turbo Transfer system (Bio-Rad) according to manufacturer’s instructions. For immunodetection of O-GlcNAc modified proteins, western-blot was performed with CTD110.6 antibody (1:5,000 dilution, Sigma-Aldrich) as already published^{28 (link)}. Phosphorylated proteins were detected using anti-phospho-Threonine antibodies (Q7 at 1:500, Qiagen) according to manufacturer’s protocol (Phosphoprotein Purification kit, Qiagen).

First tissue of the left ventricle was homogenized by using RIPA Lysis and Extraction Buffer (ThermoFisher Scientific, Waltham, MA, USA) as well as Phenylmethanesulfonyl fluoride, Protease Inhibitor Cocktail and Sodium orthovanadate (all three from Sigma-Aldrich, St. Louis, MO, USA). With Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) the protein concentration in homogenates was measured. For electrophoresis the homogenates were loaded onto a 4–20% Mini-PROTEAN^® TGX Stain-Free™ Protein Gel (BioRad, Hercules, CA, USA) and afterwards transferred with a Trans-Blot^® Turbo™ Transfer System using Mini PVDF Transfer Packs (both from Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% milk for 1,5 h at room temperature and afterwards incubated with the respective primary antibody for connexin 43 (Cx43) (CellSignaling Technology, Danvers, MA, USA) and IL-6 (abcam, Cambridge, UK) overnight at 4°C. After washing, an anti-rabbit IgG HRP-linked antibody (CellSignaling Technology, Danvers, MA, USA) was used as secondary antibody for 1 h at room temperature. The blots were analyzed with ChemiDoc (Bio-Rad, Hercules, CA, USA) and the ImageLab software (version 5.2, Bio-Rad). Results are presented as intensity.