Multi beads shocker cell disruptor

Xenograft tumours were cut into small pieces and homogenised using a Multi-Beads Shocker Cell Disruptor (Yasui Kikai, Osaka, Japan). Then, the homogenised tumour cells were lysed with cell lysis buffer [100 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor (Roche Diagnostics, Basel, Switzerland), phosphatase inhibitor (Roche Diagnostics)]. Protein concentrations were determined using a Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Then, 50 μg lysate was used for SDS-PAGE. Resolved proteins were transferred onto nitrocellulose membranes using Trans-Blot Turbo (Bio-Rad) and then membranes were probed with anti-thymidine phosphorylase (TP)(1:1000) (Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:1000) (Cell Signaling Technology). Next, membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and probed with anti-rabbit IgG-specific Alexa 680-conjugated secondary antibody (Thermo Fisher Scientific, 1:24,000), or anti-mouse IgG -specific Alexa 800-conjugated secondary antibody (Thermo Fisher Scientific, 1:24,000). Finally, membranes were washed with TBS-T and then scanned and analyzed using the Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE, USA).

S. typhimurium strain SH100 WT was used for in vivo infection experiments. S. typhimurium (1 × 10⁵ cfu/mouse) was suspended in 100 µl PBS and intraperitoneally injected. Two days post-infection, the mice were sacrificed, and the spleens were weighed. To determine colony forming unit (CFU) in organs of infected mice, spleens and livers were homogenized using a Multibeads shocker cell disruptor (YASUI KIKAI, Osaka, Japan), and serial dilutions of homogenates were prepared and plated onto LB plates. The plates were incubated at 37°C overnight, and the resulting colonies were counted.