Protein assay reagent

Cells were harvested and lysed in 1 × RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail. The protein concentrations of each extracted protein sample were measured using Bio-Rad protein assay reagents. A total of 30 micrograms of each protein sample was subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% skim milk and then incubated with specific primary antibody overnight. The antibody-probed membrane was then washed 6 times with 1 × PBST (0.05% Tween 20 in 1 × PBS). After washing, the appropriate horseradish peroxidase-labeled secondary antibodies were added to the membrane for 1 h, and then washed with 1 × PBST. The bound antibodies were detected by enhanced chemiluminescence (ECL) reagents (GE Healthcare Life Sciences; Uppsala, Sweden). The blot signals were visualized by X-ray film (Roche Applied Science, Mannheim, Germany). The intensities of signals were quantified by GeneTools software (SYNGEN, Cambridge, UK).

β-Lactamase specific activity was determined spectrophotometrically in crude sonic extracts from strain 105.5R(r) and the seven above-described ampD mutants. To determine the β-lactamase specific activity after induction, the cultures were incubated in the presence of 40 μg/ml cefoxitin for 1 h before harvesting. The samples were centrifuged for 5 min at 3000 × g and the pellets were washed once in 5 ml 0.01 M phosphate buffered saline, pH 7.0 and re-suspended in 1 ml of the same buffer. The suspensions were sonicated on ice for 3 min, and then centrifuged at 10,000 × g for 30 min at 4°C and the supernatant was retained. After determining the total protein using Bio-Rad protein assay reagents, 10 min reactions were allowed before nitrocefin hydrolysis was measured. The specific activity (U/mg) was calculated as nanomoles of nitrocefin hydrolyzed per minute per milligram of protein, using an extinction coefficient (Δε) of 20,500 M⁻¹ cm⁻¹ for nitrocefin at 486 nm, as suggested by the manufacturer (Oxoid, UK; Kong et al., 2005 (link)). The mean β-lactamase activity obtained in three independent experiments was analyzed.

Total protein was isolated from CS-1 and SW1353 cells with 1 × RIPA lysis buffer (Upstate Biotechnology, Charlottesville, VA) supplemented with complete protease inhibitor cocktail tablets (Roche Applied Science, IN, USA). Protein concentrations were examined by Protein Assay Reagents (Bio-Rad, Hercules, USA). Equal amounts of denatured proteins were separated by NuPAGE® 4–12% Bis-Tris Gel (Life Technologies) and transferred onto a nitrocellulose membrane (Bio-Rad). After blocking in 5% non-fat milk for two hours, the membranes were incubated with specific primary antibodies WIF1 (Cell Signaling Technology, catalog number: 5652, 1:1000 dilution), Wnt5a/b (Cell Signaling Technology, catalog number: 2530, 1:1000 dilution), LRP6 (Cell Signaling Technology, catalog number: 3395, 1:1000 dilution), Dvl2 (Cell Signaling Technology, catalog number: 3224, 1:1000 dilution), and β-Actin (Sigma-Aldrich, dilution 1:2000) at 4 °C overnight. The membranes were further probed with respective secondary antibodies (LI-COR Biosciences, NE, USA), and scanned by Odyssey® CLx equipment (LI-COR Biosciences, NE, USA) to detect the bands and the density.