Optitran ba s 83

3 × 10⁶ BMDMs were infected with L. monocytogenes at MOI 10 as described above, or treated with 250 IU/ml of IFNβ (PBL interferon source) or 5 ng/ml of IFNγ (Affymetrix, eBioscience). For whole‐cell lysates, BMDMs were lysed in 80 μl of Frackelton buffer (10 mM Tris, 30 mM Na₄P₂O₇, 50 mM NaCl, 50 mM NaF, 1% Triton X‐100, 0.1 mM PMSF, 1 mM DTT, 0.1 mM Na₃VO₄, pH 7.5) supplemented with complete protease inhibitors diluted 1:100 (Roche). Proteins were separated on 10% SDS–polyacrylamide gels and blotted onto cellulose membranes (Optitran BA‐S 83, GE Healthcare Life Sciences) using a standard semidry protocol (1.5 h, 32 mA per gel). For tissue Western blots, spleens were lysed in 1 ml Frackelton buffer and livers were lysed in five volumes of buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% Triton X‐100, 0.1% SDS, 5 mM EDTA, 1 mM EGDA, inhibitors). The following primary antibodies were used: STAT1 C‐terminal 56, STAT1 (clone E‐23, Santa Cruz), phospho‐Y701 STAT1 (Cell Signaling), phospho‐Y689 STAT2 (Millipore), STAT2 (Millipore), STAT3 (Cell Signaling), phospho‐Y705 STAT3 (Cell Signaling), phospho‐Y694 STAT5 (BD Biosciences), STAT5 (Millipore) and tubulin (Sigma). Secondary antibodies were purchased from Li‐COR and blots were detected on Odyssey CLx^® Infrared Imaging System (Li‐COR).

Eight micrograms of mitochondrial protein were supplemented with Laemmly loading buffer and separated on 10-comb precast 10% [w/v] to 20% [w/v] acrylamide gradient gels (Life Technologies, Darmstadt, Germany). Proteins were transferred to reinforced nitrocellulose membranes (Optitran BA-S 83, GE Healthcare, Freiburg, Germany) using semi-dry blotting (Trans-Blot SD Cell, Biorad, Munich, Germany) for 90 min in transfer buffer [37 mM glycine, 130 mM tris(hydroxymethyl)aminomethane, 0.275% [w/v] SDS, 20% [v/v] methanol]. For cytochrome c quantitation monoclonal antibodies directed against pigeon cytochrome c (7H8.2C12, Pharmingen, San Diego, CA, USA) in a 1:1000 dilution were used to probe the western blots. Signal generation was then achieved using anti-mouse secondary antibodies conjugated with horseradish peroxidase (HRP) via chemiluminescence in a buffer containing 100 mM Tris pH 8.5, 0.23 mM p-coumaric acid, 1.25 mM luminol, 0.00015% [v/v] H₂O₂. Quantitation of signals from three biological replicates was performed using the ‘image J’ software. For SDH1-1 quantitation, polyclonal antibodies (Peters et al., 2012 (link)) in a 1:1000 dilution were used and subsequently detected via biotin-conjugated goat-anti-rabbit secondary antibodies. Chemiluminescence signals of three biological replicates were generated by incubation with avidin-conjugated HRP via as described above.