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Caspase activity Caspase-3 and caspase-6 activities were analysed as described previously.16 Briefly, the gastrocnemius muscle was homogenized in ice-cold buffer. The homogenate was frozen on dry ice/ethanol and heated to 37��C for three cycles. After centrifugation (20?000?g for 30?min), 100??g protein was pre-incubated in assay buffer (100?mM 4-(2-hydroxyethyl)-1-piperanzineethanesulfonic acid (HEPES) pH?7.5, 10% sucrose, 0.1% 3-(3-cholamidepropyl)dimethylammonio-1-propanesulphonate (CHAPS), 2% dimethyl sulphoxide (DMSO), and 10?mM dithiothreitol (DTT) with or without 50?��M caspase-3 or caspase-6 inhibitor Ac-DEVD-CHO or Ac-VEID-CHO) at 37��C for 30?min. The fluorogenic substrate (50?��M) Ac-DEVD-AMC or Ac-VEID-AMC was added for caspase-3 and caspase-6. Assay conditions were identical to proteasome assay. Western blotting Protein lysates were prepared from the INPP5D gastrocnemius AZD4547 mouse muscle according to standard protocols. We used primary antibodies against Akt (9272), pAkt (Ser473; 4051), pAkt (Thr308; 9275), p-p70S6K (Thr389; 9025), 4E-BP1 (53H11; 9644), p4E-BP1 (Ser65; 9451), and p4E-BP1 (Thr 37/47; 9459), all from Cell Signaling, and p70S6K (sc-230; Santa Cruz), as well as appropriate secondary antibodies. Statistics Data were analysed with GraphPad PRISM 5.0 (GraphPad Software, Inc., La Jolla, CA, USA).17 Results are shown as mean?��?standard error of mean. For the comparisons among groups, data were analysed with analysis of variance followed by post hoc comparisons using Tukey's test. Survival was assessed by Cox proportional hazard analysis. A P-value of ?0.4). At the end of the study, total tumour cell numbers were 4.20?��?0.29?��?109 cells in the placebo-treated rats and 4.19?��?0.40?��?109 cells in rats with febuxostat (P?>?0.9). There were no observations of other malignancies during necropsy. Body weight and body composition Average loss of body weight from baseline to the end of study was higher in the placebo group Cisplatin cost than in treated rats (?50.2?��?2.1?g vs. ?26.3?��?12.4?g, P?
