Soniprep 150 sonicator

Cells were seeded onto 6-well plates (Corning) and allowed to adhere overnight. After treatment, cells were lysed with ice-cold lysis buffer (50 mM Tris-base, 40 mM sodium pyrophosphate,100 μM sodium fluoride, 150 mM sodium chloride, 1% Triton X-100, 10 mM EGTA and EDTA sodium salt, 1x complete EDTA-free protease inhibitor, 1× phostop (Roche, Hertfordshire, UK). Lysates were subjected to sonication at an amplitude of 15 μm using a soniprep-150 sonicator (Sanyo, Watford, UK). Protein was quantified using a BCA assay kit and Nanodrop 2000 (both Thermo Fisher, Waltham, Massachusetts, USA).
Anti-PFKFB3, GAPDH and anti-PARP monoclonal rabbit antibodies were used at a concentration of 1:1000 for probing (#13123, #5174 and #9542S respectively Cell Signaling Technology). Mouse anti-PMCA4 (JA9, # MA1–914, Thermofisher) was also used 1:1000. HRP conjugated goat anti-rabbit antibody (#7074 Cell Signalling Technology) or HRP conjugated horse anti-mouse (#7076 Cell Signalling Technology) were used as a secondary antibody. Clarity ECL, a Chemidock and ImageLab acquisition software were used to produce digital images (BioRad). ImageJ software was used to quantify an PARP:cleaved-PARP band volumes in MIA PaCa-2, BxPC-3 and HPSCs after 6 h of treatment with 10 μM PFK15.

Cultures of SoluBL21 containing pBAD/myc-His toxin constructs were grown to an OD₆₀₀ of 0.6–0.7 prior to addition of 0.02% (w/v) l-arabinose for 3 h. Harvested cells were lysed by sonication in binding buffer containing 20 mM Tris–HCl pH 7.0, 200 mM NaCl, 5 mM imidazole and 1X protease inhibitors (Roche) using a Soniprep 150 sonicator (Sanyo). Lysates were centrifuged at 10,000×g, 0.45 µm-filtered, then incubated with TALON metal affinity resin (Clontech) for 1 h at 4 °C, prior to loading in gravity-flow columns. His-tagged proteins were eluted using binding buffer containing 300 mM imidazole. Elution fractions were immediately concentrated and buffer exchanged into storage buffer (25 mM Tris–HCl pH 7.5) using Vivaspin centrifugal concentrators (Sartorius Ltd) and stored at − 80 °C. Protein purity was estimated to be > 95% for all purified proteins by SDS-PAGE and protein concentrations determined using the BCA assay (Pierce).

A selection of eight representative UTI-related Gram-positive and Gram-negative bacteria was tested using the same HEK-293-TLR2 transfection system to determine their capacity to stimulate TLR2. All bacteria examined were of hazard group 2 or lower and were studied in a class 2 containment facility. A whole bacterial cell lysate was also prepared from H. pylori (SS1 strain) for assay in the same system. To prepare the lysate, H. pylori cultures from 10 blood agar base 2 plates (Oxoid) cultured for 24 h under microaerobic conditions at 37°C were harvested into ice-cold sterile phosphate-buffered saline. This suspension was then disrupted using a Soniprep 150 sonicator (SANYO, Watford, UK), with 6 × 10s bursts at amplitude of 10 µm, then aliquoted and stored at −20°C. There were approximately 6 × 10⁹ bacteria per ml before sonication, corresponding to 1 × 10⁷ bacteria per ml when diluted, and the protein content of the lysate was 13.8 mg/ml as determined using the BCA protein assay kit (Thermo). The TLR2-stimulating capacities of bacteria and lysates in these experiments are presented as NF-κB fold induction vs cells cultured in medium alone.