The bacterial cultures were centrifuged at 5,000 × g for 5 minutes. To study the effect of pH, the pelleted bacteria were re-suspended in 1 ml of fresh LB broth (control, pH7.0) or 1 ml of LB broth with pH 3.0, 5.0,
7.2, and 8.4, respectively, and shaken at 250 RPM and 37°C for additional 6 hours, and then collected. To study the effect of osmolarity, the pelleted bacteria were re-suspended in 1 ml of NaCl-free Akt inhibitor LB broth supplemented with 0, 42.5, 85, 170, 340, and 680 mM sodium chloride, respectively, and then shaken at 250 RPM and 37°C for additional 6 hour, and were collected. Regular LB broth, which contained 170 mM NaCl, was used as the control. To study the effect of butyrate, the pelleted bacteria were re-suspended in 1 ml of fresh LB broth (control) or 1 ml of LB broth containing 10 mM sodium butyrate and shaken at 250 RPM and 37°C for additional 6 hours, and then collected. To study the effect of oxygen ventilation, the pelleted bacteria were re-suspended
in 1.5 ml of fresh LB broth. One group of bacteria was shaken at 250 RPM and 37°C for additional 6 hours with good aeration (control) while another group of bacteria was transferred into 1.5 ml microcentrifuge tubes with their covers closed tightly, and incubated at 37°C without shaking for additional 6 hours. Preparation of culture supernatants and cell extracts from bacterial grownin vitrounder different conditions To prepare protein samples from the pellets Selleckchem PCI-32765 of bacterial cultures, the cultures (1 ml) were centrifuged at 5,000 × g and 4°C check details for 10 minutes. The pellets were re-suspended in 200 μl of bacterial lysis buffer (8 M urea, 2% chaps, and 10 mM Tris, pH8.0). The bacterial suspension was sonicated for 15 seconds three times with
an interval of 30 seconds, centrifuged at 5,000 × g and 4°C for 10 minutes, and then transferred into new tubes for Western analysis. To prepare secreted protein samples, 0.5 ml of ice-pre-cooled 25% TCA was added into the supernatants of the bacterial cultures (1 ml). The mixture was incubated at 4°C for 15 minutes, and then centrifuged at 15,000 × g and 4°C for 10 minutes to precipitate soluble proteins. The pellets were washed with acetone twice, dried in air for 30 minutes, and then re-suspended in phosphate buffered saline (PBS) for Western analysis [45,48]. The protein concentrations of the pellet and soluble proteins were determined by Bradford Method on a micro-plate reader with absorbance at 495 nm using a standard curve of BSA concentrations. In vivostudies Female BALB/c and SCID mice (6–8 weeks old) were obtained from Jackson Laboratory (Bar Harbor, ME). Mice were kept in sterilized, filter-topped cages, handled in laminar hoods, and fed autoclaved food and water under specific pathogen-free (SPF) conditions at our animal facilities.