Bicinchoninic acid method

Protein extraction was carried out as described previously (Böhme et al., 2010a (link), b (link); Carrera et al., 2017 (link)). Briefly, a loop full of each bacterial culture was harvested, and the biomass was mixed with 100 μL of a solution containing 50% acetonitrile (50% ACN) (Merck, Darmstadt, Germany) and 1% aqueous trifluoroacetic acid (1% TFA) (Acros Organics, NJ, United States). After vortexing and centrifugation, the supernatant was treated with a solution of lysis buffer [60 mM Tris-HCl pH 7.5, 1% lauryl maltoside, 5 mM phenylmethanesulfonyl fluoride (5 mM PMSF) and 1% dithiothreitol (1% DTT)]. The supernatants were transferred to a new tube and then quantified using the bicinchoninic acid method (Sigma Chemical Co., United States). All analyses were performed in triplicate.

Cells were incubated in lysis buffer (50 mM Tris-Cl buffer pH 7.5 containing 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors) for 20 min on ice. Nuclei were pelleted by centrifugation. The protein concentration in post-nuclear supernatants was determined by the bicinchoninic acid method as recommended by the manufacturer (Sigma), using bovine serum albumin as standard. Proteins from cell lysates or virus purification fractions were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Hybond-ECL; Amersham) by using a Trans-Blot apparatus (Bio-Rad). The proteins of interest were revealed with specific primary antibodies, followed by species-specific secondary antibodies conjugated to horseradish peroxidase (Jackson Immunoresearch), and enhanced chemiluminescence detection as recommended by the manufacturer (Thermofischer).

Nuclear factors were isolated using the procedure reported earlier [16] (link). Briefly, 1×10⁶ THP-1 macrophages were lysed using 300 µl of buffer A (10 mM HEPES–KOH, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF), centrifuged for 10 s at 15,000 g and the supernatants were labeled as cytosolic fraction. The cell pellet was resuspended in 200 µl of icecold buffer B (20 mM HEPES–KOH, pH 7.9, 25% glycerol, 420 nM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF), centrifuged at 15,000 g for 10 s and the supernatants were stored at −80°C until use. The concentration of proteins was quantified using bicinchoninic acid method (Sigma).
Trans-AM ATF-2, c-JUN and NF-kB kit from Active Motif (Carlsbad, CA) were used to determine the levels of ATF-2, c-JUN and p65NF-kB in nuclear extract. 2 µg of nuclear extract was added to wells coated with oligonucleotides containing the consensus binding site for the respective nuclear factors, followed by addition of primary antibody, horseradish peroxidase-conjugated secondary antibody and the substrate. The absorbance was read at 450 nm (with a reference wavelength at 650 nm). The specificity of the assay was monitored using competitive binding of wild-type or mutated consensus oligonucleotides before the addition of nuclear extracts.

Antibodies used were rabbit anti-CROCC (1:250–1:750 overnight; Novus Biologicals, 80820), rabbit anti-CROCC (1:250–1:750 overnight; Novus Biologicals, 80821), and mouse monoclonal beta-Actin (1:10000 1 hour at room temperature; Sigma-Aldrich). Cells were lysed for 20 minutes on ice in RIPA buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1% NP40, 0.5 M sodium deoxycholate, 0.1% SDS, complete protease inhibitor cocktail, PhosSTOP [Roche]). Protein concentration was quantitated using the bicinchoninic acid method (Sigma-Aldrich). Whole-cell extracts were separated by electrophoresis on a 3% to 8% Tris-Acetate gel and transferred to PVDF membrane using the iBlot2 system (ThermoFisher Scientific) according to the manufacturer’s instructions. Membranes were blocked in 5% milk dissolved in 0.1% Tween/TBS.

Protein extraction was carried out as described previously [19 (link),84 (link)]. In brief, an inoculation loop full of bacterial culture was harvested and the cells resuspended in 100 μL of a solution containing 50% acetonitrile (ACN; Merck, Darmstadt, Germany) and 1% aqueous trifluoroacetic acid (TFA; Acros Organics, NJ, US). After vortexing and centrifuging, the supernatant was treated with a solution of lysis buffer containing 60 mM Tris-HCl pH 7.5, 1% lauryl maltoside, 5 mM phenylmethanesulfonyl fluoride (PMSF) (Sigma, St. Louis, MO, US), and 1% dithiothreitol (DTT) (Sigma Chemical Co., US). The supernatant was then transferred to a fresh tube and the amount of protein was determined by the bicinchoninic acid method (Sigma Chemical Co., US). All experiments were performed in triplicates.