Clarity max western ecl blotting substrate

Total proteins were extracted using SDS lysis buffer (4 %w/v SDS; 20 %v/v glycerol; 0.004 %w/v bromophenol blue; 0.125 M Tris-Cl, pH 6.8; 10 %v/v 2-mercaptoethanol) and sonication (50 kHz for 30 s; VibraCell X130PB, Sonics Materials) at 4 °C and subsequently denatured at 95 °C for 5 min. Proteins were separated on RunBlue 4-12 %w/v bis-tris polyacrylamide gels (Expedeon) and then transferred onto iBlot PVDF membranes (ThermoFisher) using the Wet/Tank Blotting Systems (Bio-Rad). Membranes were probed overnight at 4 °C in blocking solution containing the primary antibody. Primary antibodies used in this work were PARP14 (C-1) (Santa Cruz Biotechnology, sc-377150), Phospho-STAT1 (pSTAT1; Tyr701; 58D6) (Cell Signalling Technology, 9167 L), STAT1 (Cell Signalling Technology, 9172), MHC Class I H2 Kb (Abcam, ab93364), GAPDH (Proteintech, 60004-1-Ig), TAP1 (Cell Signalling Technology, 12341), TAP2 (Cell Signalling Technology, 12259; Santa Cruz Biotechnology, sc-515576), and PD-L1/B7-H1 (R&D Systems, AF1019-SP; Cell Signalling Technology, 13684). This was followed by incubation with the appropriate secondary antibody for 1.5 h at room temperature. Signals were developed using the Clarity Max Western ECL blotting substrate (Bio-Rad) and acquired on a Gel Doc XR+ Gel Documentation System (Bio-Rad). Images were analysed using Image Lab™ Software (version 3.0.1.).

In order to determine the presence or absence of myophosphorylase, a Western blot assay was performed. At days 7 and 14 of the skeletal muscle final differentiation, cultures were scrapped and centrifuged at 300× g 5 min. Protein extraction was performed by adding a cell lysis buffer (Tris-HCl mM pH 7,5, NaCl 150 mM, EDTA 5 mM and SDS 0.1%) with protease inhibitors (Roche, Basel, Switzerland, 11873580001). Protein quantification was accomplished using a DC^TM protein assay kit, following the instructions of the manufacturer (BioRad, Hercules, CA, USA, 5000112). Subsequently, we performed an SDS-PAGE in 4–20% Mini-PROTEAN^® TGX™ precast protein gels (Bio-Rad, USA, #4561094) and transferred the proteins to a nitrocellulose membrane using the Trans-Blot Turbo Transfer system (BioRad, USA, 1704158). After incubation with primary antibodies for 17 h at 4 °C, we incubated the membranes with the GARPO secondary antibody (Molecular Probes, USA, G21234) 1:2500 for 1 h at room temperature. Protein detection was performed with a Clarity Max™ Western ECL Blotting Substrate (BioRad, USA, 1705062). α-tubulin-HRP (Abcam, UK, ab40742) was used as a loading control at 1:5000 dilution.

Lung samples were homogenized with a tissue lyser (Qiagen, Hilden, Germany), proteins were isolated using the NucleoSpin® RNA/Protein Kit (Macherey-Nagel, Düren, Germany) and protein concentration was measured with a protein quantification assay (Macherey-Nagel).
20 µg proteins per lane were loaded, fractionated by SDS-PAGE (polyacrylamide gel concentrations in Table 1) and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked (blocking conditions in Table 1), incubated with the primary antibody, washed, incubated with the secondary antibody (antibody details in Table 1), washed and developed using the Clarity Max™ Western ECL Blotting Substrate (Bio-Rad) and the ChemiDocTM Touch Imager (Bio-Rad). Protein bands intensities were assessed with the Image LabTM Software (Bio-Rad), normalized according to the loading control and expressed as percentage of the CD mean of the respective membrane.