Pefabloc sc

Cultured MEF cells were washed with PBS 3 times. The following steps were carried out either on ice or at 4°C. Cells collected were suspended with 10 mM Tris-HCl buffer (pH7.5) containing 1mM ethylenediaminetetraacetic acid (EDTA), 250 mM sucrose, 1 mM dithiothreitol (DTT), 1 mM AEBSF (Pefabloc SC, Roche) and various protease inhibitors (1 × complete protease inhibitor cocktail (Roche)) at a density of 5 × 10⁷ cell/mL, and were homogenized using a Potter-Elvehjem homogenizer. The extracts thus obtained were cleared at 15,000 rpm for 10 min at 4°C and the supernatant was transferred to a new tube. The sample was ultracentrifuged at 100,000×g for 1 h at 4°C, and the supernatant was collected and used as the cytoplasmic fraction.

TAP was performed as by Fernández et al. (2009) (link). Briefly, mouse forebrain was homogenized on ice in 1% deoxycholate (DOC) buffer (50 mM Tris [pH 9.0], 1% sodium deoxycholate, 50 mM NaF, 20 μM ZnCl₂, and 1 mM Na₃VO₄), 2 mM Pefabloc SC (Roche), and 1 tablet/10 mL protease inhibitor cocktail tablets (Roche) at 0.38 g wet weight per 7 mL cold buffer with a glass Teflon Douncer homogenizer. The homogenate was incubated for 1 hr at 4°C and clarified at 50,000 × g for 30 min at 4°C. TAP-tagged complexes were isolated as described previously (Fernández et al., 2009 (link)). The SDS-PAGE gel was fixed and stained with colloidal Coomassie, and lanes were cut into slices, destained, and digested overnight with trypsin (Roche, trypsin modified, sequencing grade) as described previously (Fernández et al., 2009 (link)). Peptide digestion, LC-MS/MS, and proteomics data analysis are described in the Supplemental Experimental Procedures.

One day before the first visit, the enrolled patients were confined in the hospital and served the same meals for lunch and dinner. Faeces and urine were collected in dispensable sterile containers. Samples were kept on dry ice before being stored at −80°C. Patients were fasted overnight. At 0800 hours, the patients ingested a carbohydrate meal containing 100 g of flour as an oral glucose load^{24 (link)}. Blood samples were taken before and 30 min, 60 min, 120 min and 180 min after the meal test. The blood samples were immediately supplemented with a dipeptidyl peptidase-4 inhibitor (for GLP-1 measurement; Millipore, Billerica, MA, USA), the serine protease inhibitor Pefabloc SC (AEBSF for active ghrelin measurement; Roche, Basel, Switzerland) and a protease inhibitor cocktail (Millipore, Billerica, MA, USA). Serum and plasma samples were prepared and kept on dry ice before storage at −80°C.

A Saponin extract of MLN from Salmonella-infected mice at day 6 p.i. was made by collecting MLN in 0.05% PBS-Tween containing Pefabloc SC (Roche), soybean trypsin inhibitor, bovine serum albumin and EDTA (all from Sigma). MLN were weighed, Saponin (Sigma) was added to a final concentration of 2% (w/v) in PBS and samples were incubated overnight at 4°C. The samples were then centrifuged at 13,000 × g for 10 min and supernatants were collected and stored at −20°C until analysis. IL-12p70 was quantitated using the Bio-Plex Pro Mouse Cytokine IL-12p70 set from Bio-Rad (Hercules, CA) according to manufacturer's recommendation. The data was acquired in a MAGPIX instrument from Luminex (Austin, TX) and analyzed with the xPONENT software by Luminex.

Recombinant TorZ (rTorZ) was purified from 4 L of protein expression culture. The cell pellets were resuspended in 20 mM Tris-Cl pH 8.0, 5% glycerol, 1 mM Pefabloc SC (Roche) (∼5 mL/g wet weight) and lysed by three passes through a French Press (Aminco) at 15000 psi. Imidazole was added to a concentration of 20 mM, followed by removal of cell debris by centrifugation at 30 000 × g, 4°C, 30 min. The resulting cell-free extract and was loaded onto a 5 mL HiTrap Ni-NTA Sepharose column (GE Healthcare Biosciences) equilibrated with 20 mM K-Phosphate pH 7.4, 500 mM NaCl, 20 mM imidazole (buffer A). The elution buffer (buffer B) was the same as buffer A but contained 500 mM imidazole. Following loading of the sample, the column was washed with 5 CV of buffer A, followed by a linear gradient (0–100% buffer B) over 10 and 2 CV of 100% buffer B. SDS-PAGE was used to assess the purity of fractions, followed by pooling according to rTorZ purity. Protein pools were concentrated and separated on a Superdex 200 HiLoad (16/60) gel filtration column (GE Healthcare Biosciences) equilibrated in 20 mM Tris-Cl pH 7.8, 150 mM NaCl, 5% glycerol. Separated proteins were again pooled according to purity, concentrated and desalted using a PD-10 column (GE Healthcare Biosciences) equilibrated in 20 mM Tris-Cl pH 7.8 5% glycerol. Purified proteins were stored at -80°C until further use.