Cell lytic mt buffer

For each ATP and protein measurement a total of 3 co-cultured slices were placed in 400 µL Cell Lytic MT buffer (Sigma, Zwijndrecht, the Netherlands). These were homogenized twice (15 sec, 6500 g, 8°C) using a tissue homogenizer Precellys 24 Bertin Technologies (Labmakelaar Benelux B.V. Rotterdam, The Netherlands). To remove cellular debris, the homogenates were centrifuged for 5 min (14000 g, 8°C) and the remaining supernatant was divided into 2 portions of 200 µL. One portion was stored at −80°C for protein measurement and the second 200 µL portion was mixed with 100 µL of ATP lytic buffer from ATPlite kit (Perkin Elmer, Oosterhout, The Netherlands) for ATP measurement, which was carried out with a microplate reader Synergy TM HT Multi Detection Microplate Reader (Biotek Instruments Inc, Abcoude, the Netherlands) with settings for luminescence: 590/635 nm, top measurement, and sensitivity 230. ATP was determined in technical duplicates and luminescence values were recalculated as µM ATP in total liver slice extracts.
Protein concentration was determined by the Bradford method protein assay (BioRad, Veenendaal, The Netherlands). Protein samples of 2 µL were diluted 80 times in PBS and measured, with BSA used as a standard, each measurement being taken in duplicate. ATP concentration was normalized to mg of protein per slice.

Small intestinal samples were homogenized in Cell Lytic MT buffer (Sigma) using Lysing Matrix E beads (MP Biomedicals) at a concentration of 50mg/ml. Homogenates were then cleared by centrifugation at 10 000 rpm for 15 minutes before quantification of protein using BCA kit (Pierce). About 250 mg of total protein was then used to probe Murine antibody inflammation arrays (Abcam; ab133999) as per manufacturer's instructions. In brief, membranes were for 30 minutes before application of homogenized whole small intestinal lysates in blocking buffer overnight at 4°C. Following washing, biotin conjugated anti cytokine antibodies were applied for 2 hours before further washing and application of HRP conjugated streptavidin. Chemiluminescent detection was then performed using supplied detection reagents and visualization performed using FluorChem E imaging system (Protein Simple). See Table S1 for plate layout.

Muscle tissue was homogenized using a Bullet Blender (Next Advance, NY USA) in CellLytic MT buffer (Sigma Aldrich) using 0.5 mm Zirconium oxide beads. The supernatant was aliquoted and utilized for Bradford assay (Beckman Coulter DU 730 spectrophotometer at 595 nm) to determine total protein content. Standard Western Blot protocol (Abcam) was performed using 8 ul of sample and ladder (Bio-Rad) loaded into SDS gel wells (ClearPAGE; 4–20%), run in a Tris-Tricine SDS running buffer (ClearPage) and transferred using Tris/Glycine buffer (Bio-Rad). Three primary antibodies specific to the myosin heavy chains I, IIa and IIb (Developmental Studies Hybridoma Bank, University of Iowa, BA-D5 (1:750), SC-71 (1:500) and BF-F3 (1:1) respectively) and two secondary antibodies [KPL peroxidase labeled goat anti-mouse IgG (H+L) at 1:10,000, Invitrogen HRP goat anti-mouse IgG+A+M at 1:30,000] were used. Primary antibodies have been previously confirmed for fiber typing in pinnipeds (Watson et al., 2003 (link); Kanatous et al., 2008 (link)). Protein bands on a nitrocellulous membrane were visualized using a chemiluminescent substrate kit (KPL International) sensitive to peroxidase-labeled antibodies and developed using a luminescent image analyzer (GE LAS 4000) and accompanying software (GE Healthcare Life Sciences). Western Blot analysis was completed in duplicate.