Precellys 24

Cryogenically powdered tumour samples from human tumour xenograft models were purchased from Charles River Discovery Research Services. Cell lines (to generate xenograft models) were obtained from ATCC or NCI and authenticated by STR profiling. 20–30 mg of tumour was homogenized in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche) using a Precellys 24 homogeniser and centrifuged twice at 4 °C for 10 minutes at 13 000 rpm. Protein concentration was determined using a BCA kit (Pierce).

Tissues were prepared as previously described^{29 (link)}. Briefly, the tissues were dissected into small pieces and homogenized at 4 °C using the Precellys 24 homogenizer in PBS with protease and phosphatase inhibitors (Roche,Indianapolis, Indiana). For fractionation, brain tissue was homogenized in a teflon dounce grinder on ice in PBS with protease and phosphatase inhibitors (Roche, Indianapolis, Indiana). After homogenization, protein concentration was determined with a Pierce BCA protein assay (Life Technologies, Grand Island, NY).

The protein levels of PGLYRP2, IL1β, IL6, and TNFα were measured in jejunal samples. One centimeter jejunal tissue specimens (weight, 350–500 mg, stored at −80°C) were homogenized using a Precellys 24 homogenizer in 800 μl PBS containing EDTA-free protease inhibitor (Roche Applied Science, Penzberg, Germany). Homogenized tissue supernatants were standardized to 1 mg/ml after protein concentration measurements using the DC protein assay according to the manufacturer's recommendations (Bio-Rad Laboratories, Hercules, CA, USA). Measurements of IL1β, IL6, and TNFα by ELISA were performed using DuoSet® ELISA according to the manufacturer's instructions (R&D Systems, Inc., Minneapolis, MN, USA). Measured cytokine concentrations were standardized to the initial weight of sample. We evaluated commercially available ELISA kits for PGLYRP2 (Amsbio, Abingdon, U.K.), however they turned out not to be specific for PGLYRP2 and thus cannot be trusted. Data is not shown. For details on ELISA kits see Supplementary Table 3.