Np 40

For Western blot analysis, half tibialis anterior muscles were snap-frozen, lysed using lysis buffer [50 mM Tris-HCl pH 7.8, 150 mM NaCl, 1% NP-40 (AppliChem/A1694), 10% glycerol, 5 mM EDTA, 1 mM EGTA, 1 Halt Protease and Phosphatase Single-Use Inhibitor Cocktail (FisherScientific/10025743), and 0.5 mM PMSF (AppliChem/A0999), pH adjusted to 7.4], and subjected to SDS-PAGE followed by Western blot analysis as already described (Wild et al., 2016 (link); Straka et al., 2018 (link)). In each lane, equal amounts of protein were loaded (20 μg). Chemiluminescence signals were obtained using an ECL system (Biozym Scientific GmbH/541004) in combination with a Syngene G:Box Chemi XX6 chemiluminescence imager (Thermo Fisher Scientific, Schwerte, Germany). The analysis used ImageJ freeware image processing software².

Proteins were extracted using Nonidet-P40 lysis buffer (150 mM sodium chloride, 50 mM Tris [pH 8.0], 1% NP-40 (Applichem), and Roche cOmplete Protease Inhibitor Cocktail). After 15 minutes of centrifugation at 14,000g at 4°C, the protein concentration was determined with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). In total, 10 μg or 20 μg of proteins were separated by electrophoresis in SDS polyacrylamide gel. The proteins were then transferred into the nitrocellulose membrane Macherey-Nagel NCP Porablot Membrane and incubated for 1 hour at room temperature in 3% BSA in PBS-Tween (0.01%) buffer. Next, the membrane was incubated overnight at 4°C with the antibodies listed in the Supplemental Table 3 with agitation. All the loadings were controlled with Ponceau S staining and anti-GAPDH antibody. When the primary antibodies are fixed, the membrane were rinsed with PBS-Tween (0.01%) buffer and followed by horseradish peroxidase–coupled (HRP-coupled) secondary antibody incubation. Proteins were finally revealed by detecting of HRP with the SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific). The chemiluminescent signal was captured by the Syngene PXi image analysis system and analyzed using Quantity One Analyses Software (Bio-Rad).

Protein concentrations were determined using Bio-Rad DC™ Protein Assay (Bio-Rad laboratories, Hercules, CA, USA). A total of 30 to 35 µg of total protein were separated on a 10% or 12% SDS gel and afterwards transferred onto a nitrocellulose membrane by semi-dry blotting. Membranes were blocked with blocking buffer (5% BSA [Carl Roth GmbH + Co. KG, Karlsruhe, Germany] in T-BST and 1% NP 40 [Appli Chem GmbH, Darmstadt, Germany]), washed three times with washing buffer (TBST: 100 mL TBS 10×, 900 mL aqua dest., 1 mL Tween 20 [Carl Roth GmbH + Co. KG, Karlsruhe, Germany]) and incubated overnight at 4 °C with antibodies against NF-κB p65, pNF-κB p65, IκBα, pIκBα, STAT3, pSTAT3 or GAPDH diluted in 5% BSA in TBST following the instructions of the manufacturer. After three washing steps with TBST, HRP-conjugated secondary antibodies, diluted in 5% milk powder in TBST, were added to the membranes and incubated for 1 h at room temperature. Following three washing steps and incubation with Immobilon Western HRP Substrate (EMD Millipore, Darmstadt, Germany), detection of chemiluminescent blots was achieved using a western blot imaging system called the Fluor Chem E System (Protein Simple, San Jose, CA, USA). Protein bands were visualized and quantified using Image Studio version 5.2 (LI-COR Biosciences, Lincoln, NE, USA).

A density gradient was prepared in Ultra-Clear tubes (14 × 89 mm, Beckman Coulter, Brea, CA, USA) ranging from 0 to 48% (w/w) OptiPrep Density Gradient Medium (Sigma-Aldrich) in TBS buffer supplemented with 0.5% (w/v) BSA. Cell culture SN treated with or without 1% NP40 (Applichem, Darmstadt, Deutschland) was layered onto the gradient and then centrifuged using a SW-41 Ti rotor (Beckman Coulter) at 200,000× g at 5 °C for 6 h in an Optima XPN-80 ultracentrifuge (Beckman Coulter). Fractions of 340 µL were collected from the top. The density of each fraction was measured by refractometry, and the RNA concentration was quantitated by one-step RT-PCR after RNA extraction.