Bradford or bca assay

After removing the insoluble fractions as described above under “Preparation of Hp culture filtrate (HPCF)”, the VacA-containing soluble fraction was loaded into a column packed with anion exchange resin (DEAE Sephacel, GE Healthcare) that had been pre-equilibrated with wash buffer (10 mM sodium phosphate dibasic, pH 7.0). After washing the column with 3 bed volumes of wash buffer, VacA was eluted with wash buffer supplemented with 0.2 M NaCl, and collected in 1 mL fractions. After evaluating the purity of VacA within the fractions by SDS-PAGE gel separation and staining with G-250 Coomassie Brilliant Blue (Sigma-Aldrich), the fractions containing VacA with no additional visible bands were combined and concentrated using a 10 kDa MWCO centrifugal filter (Amicon Ultra, Millipore), and then dialyzed for 12 h at 4 °C in PBS pH 7.4 (at 100 times the volume of the concentrate) with stirring using a 10 kDa MWCO dialysis cassette (Slide-A-Lyzer). The concentration of purified VacA was determined by the Bradford or BCA assay (both from Thermo Fisher Scientific).
Just prior to addition to cultured host cells or tissue, VacA was activated by mixing 10% (v/v) HCl (0.3 M) to purified toxin, and then incubating at 37 °C. After 30 min, the solution was neutralized by adding NaOH (0.3 M) at an equivalent volume as the HCl added.

AKR-2B mouse embryo fibroblasts (MEFs) were seeded at 4.0 × 10⁴ in McCoy’s 5A medium supplemented with 5% heat-inactivated FBS in 6-well plates. Cells were incubated at 37°C in an incubator with 5% CO₂ for 24 h to facilitate adherence to the plate. MEFs were transiently transfected with a total of 2 μg/mL of DNA consisting of 0.1 μg/mL of pSV40-βGal, 1 μg/mL of expression plasmid encoding single, double, or triple point mutations, and 0.9 μg/mL of Acta2 promoter-reporter construct, pVSMP8-luciferase^{33 (link)}. After 48 h incubation at 37°C, transfected cells were washed with PBS and harvested by lysis in 1× Passive Lysis Buffer (Promega) supplemented with protease inhibitors leupeptin, pepstatin A, and aprotinin at 1.0 μg/mL. Cleared lysates were assayed for total protein content by either Bradford or BCA™ assay (Thermo Scientific) and reporter gene expression by luciferase activity assay (Promega). Datasets were analyzed by performing a one-way analysis of variance and Tukey’s multiple comparison test with significance of p <0.05 using Prism 6 (Graphpad Software, Inc.).