Pierce bca

The A. hydrophila ATCC^® 7966^TM strain was identified by the multilocus phylogenetic analysis (MLPA) proposed by Martinez-Murcia et al., 2011 (link).
The OMVs purification was performed according to the protocol described by Avila-Calderón et al. (2012) (link). Briefly, A. hydrophila ATCC^® 7966^TM was cultured massively on tryptic soy agar (TSA) (Becton Dickinson-BD^TM) plates incubated 24 h at 37°C. Then bacteria were harvested with a rubber policeman and suspended in 250 ml sterile PBS 0.1 M (Gibco^®). The bacterial suspension was centrifuged at 10,000 × g for 30 min (Thermo Scientific^TM Sorvall^TM Legend^TM XT/XF.) The supernatant was passed through a 0.22 μm filter (Millipore Corp.); and a sterility test was performed by culturing an aliquot onto a TSA plate followed by incubation for 24 h at 37°C. The sterile supernatant was centrifuged at 100,000 × g for 2 h at 4°C (Beckman Coulter Optima L-90K). The pellet was washed twice with 25 mL of sterile PBS and the OMVs were suspended in 1 mL of sterile PBS (raw OMVs). The total protein concentration was determined using PIERCE-BCA (Thermo Fisher Scientific Inc.) reagents as per manufacturer’s recommendations. The OMVs samples were divided into 0.5 mL aliquots and stored at -20°C until used.

In all, 10 × 10⁶ cells per sample were treated and then lysed in buffer containing 20 mM Tris pH 7.4, 150 mM sodium chloride, 10% glycerol, 0.2% NP-40, 0.1 mM sodium orthovandate, 5 mM glycerophosphate and Protease Inhibitor Cocktail Set V for 20 min on ice. Lysates were cleared by centrifugation at 10,000 × g at 4 °C and protein concentration measured using Pierce BCA (ThermoFisher Scientific). Equivalent amount of protein in each sample were immunoprecipitated by rotating with 0.25–1 µg of FADD antibody overnight at 4 °C. Immune complexes were precipitated with Protein A/G beads. After extensive washing, the beads were eluted with SDS-sample buffer at 70 °C for 20 min. Sequential blotting with anti-RIPK1 and anti-FADD was carried out subsequently. To detect RIPK1 ubiquitination, immunoprecipitation was carried out using anti-RIPK1 followed by sequential blotting with anti-ubiquitin and anti-RIPK1.

Protein concentrations in antivenoms (GPAV, HPAV, CRMAV and SABU) were determined using Thermo Scientific™ Pierce™ BCA (bicinchoninic acid) Protein Assay Kit (Thermo Scientific™ Pierce™, Waltham, MA, USA). The protein concentrations were expressed as means ± SEM of triplicates.

Liver sections kept in Krebs’s buffer (1 ml), containing the Complete Protease Inhibitor Cocktail (Sigma), were thaw up and homogenized using a TissueRuptor (Qiagen) freshly before use. Then, samples were centrifuged (10,000 ×g/15 min) to get clear supernatants for cytokine determination. Tumor necrosis factor (TNF)-α and granulocyte-monocyte colony stimulating factor (GM-CSF) (ImmunoTools) were determined by ELISAs according to the manufacturer’s instructions. Different aliquots of liver sections were homogenized prior to quantification of the total phospholipids content using a commercial ELISA kit (Abcam). The results of the ELISA assays were normalized according to the total protein content (Pierce^®-BCA, Thermo Scientific™).

Whole-cell protein lysates of A549 cells or EMT-induced A549 cells were prepared in RIPA lysis buffer containing protease inhibitors. Pierce BCA (Thermo Scientific) assay was used to measure the protein concentrations of cell lysates. Protein samples (20 μg for each) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene membranes. The membranes were then blocked with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST) solution. The blots were incubated with the same anti-E-cadherin and anti-vimentin antibodies as described above in blocking solution at 4 °C overnight. After rinsing with TBST buffer, the blots were incubated with secondary antibodies of the corresponding species conjugated with horseradish peroxidase (Beyotime Biotechnology). After incubation for 1 h, the blots were rinsed with TBST buffer and developed with an enhanced chemiluminescence detection system (Tanon).