Irdye 680 and 800

Western blots were performed to assess protein levels of Kir6.1 and GAPDH. In brief, mouse lungs with and without 18 h endotoxemia (three independent experiments with n = 3 each) were homogenized at 4 °C in PBS with 5 mM EGTA and protease inhibitor mix (Roche Diagnostics GmbH, Germany) and centrifuged at 10,000 g and 4 °C for 10 min. Supernatant protein was subjected to electrophoresis, transferred to a PVDF membrane, blocked for 1 h at room temperature with i-Block 0,5% and probed with anti-Kir6.1 (1:500, Alomone, Jerusalem, Israel) and anti-GAPDH (1:10,000, Millipore, Darmstadt, Germany) overnight at 4 °C. For negative control, 1 μg of purified Kir6.1 control peptide antigen (Alomone) was preincubated with 1 μg of antibody for one hour at room temperature and then incubated with the membrane overnight at 4 °C. The PVDF membranes were then incubated with corresponding secondary antibodies (1:10,000 IRDye 680 and 800, LI-COR Biotechnology, Lincoln, USA). Proteins were visualized with a LI-COR infrared imager (Odyssey, LI-COR Biotechnology), quantitative densitometric analysis was performed by applying Odyssey version 1.2 infrared imaging software and signals were normalized to GAPDH. Coincubation with the Kir6.1 control peptide antigen faded away the Kir6.1 immunoreactive band at a size of 50 kDa.

Total protein extracts were isolated by using 10 × Cell Lysis Buffer (Cell Signaling/New England Biolabs, Cambridge, UK) supplemented with 1 × Protease Inhibitor Cocktail (Sigma, St. Louis, USA). Protein concentrations were measured using the Bradford protein assay (Bio-Rad, California, USA). Thirty to 50 µg of total protein extracts were separated using 8 to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by electro-transfer of proteins to a nitrocellulose membrane. After blocking the membrane with Tris-buffered saline/Tween 20 (TBST) containing 5% milk or bovine serum albumin, primary antibodies were added and the membrane was incubated at 4°C overnight. After washing, the appropriate secondary antibodies (1:20,000; IRDye 680 and 800, LiCor Biosciences, Bad Homburg, Germany) were added and incubated at room temperature for 1 h. Signal detection and visualization were performed with the Odyssey-CLx Infrared Imaging system and the ImageStudio Lite software (LiCor Biosciences). Antibodies used for Western Immunoblotting are listed in Suppl. Table 5.