25 mM) in a microtiter plate (FlexStation II384, Molecular Device Inc, CA), in Tris-HCl buffer (25 mmol/L, pH 7.5). Kinetic analyses were carried out using 50 mg of protein for 30 min at 37uC in the absence and presence of 25 mmol/L ATP plus 5.0 mmol/L MgCl2, with the difference attributed to ATP- dependent proteasomal activity. Excitation/emission wavelengths were 350/440 nm. Data were normalized by proteasomal activity in the presence of 2 mmol/L of epoxomicin (a selective proteasome inhibitor). The purified 20S proteasome activity (Fig. 2A) was carried out after finishing the in vitro proteasome phosphorylation assay (see method for proteasome phosphorylation assay). Cellular oxidized proteins Protein oxidation was determined as previously described [45]. We evaluated oxidatively modified proteins using an Oxyblot kit (S7150, Millipore, MA). Samples were normalized by GAPDH and expressed as percent control. Statistics Data are expressed as mean 6 s.e.m. One-way analysis of variance (ANOVA) with post-hoc testing by Tukey was used to analyze data from Fig. 1b–e,i, 2a–c, 3a,c,e–g, 5b–e and 6a–c,e. Two-way repeated measures analysis of variance (ANOVA) with post-hoc testing by Tukey was used to analyze data from Fig. 3h. Student’s t-test (one-tailed distribution/two-sample equal variance) was used to analyze data from Fig. 1j and 3e. Linear regression analysis and correlation test by Pearson’s method were used to assess concordance of Fig. 1f,g and 6d. Survival was analyzed by the standard Kaplan-Meier analysis with log-rank test. A value of P,0.05 was considered significant. Reagents, peptide synthesis, electron microscopy, tissue fractionation, immunoprecipitation, immunoblot assays and human samples Detailed methods can be found in the Supporting Information S1. Supporting Information Figure S1 PKCbII inhibition improved ATP-dependent proteasomal activity and decreased hydrogen peroxide- induced accumulation of oxidized proteins and cell death in cultured neonatal cardiomyocytes. a. Schematic panel of PKCbII inhibition and hydrogen peroxide challenge in cultured neonatal cardiomyocytes. b. Cultured neonatal cardio- myocytes were pre-treated with TAT or bIIV5-3 plus/minus epoxomicin and challenged with hydrogen peroxide for 5 min. Proteasomal activity, oxidized proteins accumulation and cell death were evaluated after 24 hrs. Cell death was evaluated by lactate dehydrogenase (LDH) release assay in the medium after 24 hrs. Epoxomicin abrogated the bIIV5-3 cytoprotective effect. Error bars indicate SEM. *, p,0.05 compared to control (non- treated cells). 1, p,0.05 compared to bIIV5-3-treated cells. Data References 1. Wang X, Robbins J (2006) Heart failure and protein quality control. Circ Res 99: 1315–1328. 2. Patterson C, Ike C, Willis PWt, Stouffer GA, Willis MS (2007) The bitter end: the ubiquitin-proteasome system and cardiac dysfunction. Circulation 115: 1456–1463. 3. Liu J, Chen Q, Huang W, Horak KM, Zheng H, et al. (2006) Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts. FASEB J 20: 362–364. 4. Weekes J, Morrison K, Mullen A, Wait R, Barton P, et al. (2003) Hyperubiquitination of proteins in dilated cardiomyopathy. Proteomics 3: 208–216. PKCbII Inhibition in Heart Failure were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey. (DOC) Figure S2 Sustained treatment with bIIV5-3 decreased PKCbII translocation to active fraction in myocardial infarction-induced heart failure rats. The ratio of translo- cation of PKC to active fraction (Triton-soluble proteins or the particulate fraction/total fraction) in 22-week old rats (10 wks after MI surgery) (n = 6 per group). Total and Triton-soluble fractions were normalized against GAPDH and Gao, respectively. Error bars indicate SEM. *, p,0.05 compared to control (sham rats, trace). 1, p,0.05 compared to bIIV5-3-treated heart failure rats. Total PKC levels and translocations were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey. (DOC) Figure S3 Sustained bIIV5-3 treatment decreased a-b- crystallin, HSP27, cleaved caspase-3, p53 and IkB protein levels in myocardial infarction-induced heart failure. a. Representative blots of 20S proteasome subunits (a5/ 7, b1, b5 and b7), a-b-crystallin, HSP27, caspase-3, cleaved caspase-3, p53, IkB and GAPDH protein levels in heart samples from 22 week-old rats (10 wks after MI surgery) (n = 6 per group). b. Cardiac proteasome 20S proteasome subunits (a5/7, b1, b5 and b7), c. a-b-crystallin, d. HSP27, e. HSP 90, f. Ratio of cardiac cleaved caspase-3/caspase-3, g. p53 and h. IkB were measured in left ventricle tissue from 22 week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT- treated (gray bar), bIIV5-3-treated (green bar) and control (sham, white bar) rats. Data were normalized against GAPDH. Error bars indicate SEM. *, p,0.05 compared to control (sham). 1, p,0.05 compared to bIIV5-3-treated heart failure rats. Data were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey. (DOC) Table S1 Individual characteristics of left ventricular remodeling and heart failure patients. (DOC) Supporting Information S1 Reagents, peptide synthesis, electron microscopy, tissue fractionation, immunoprecipitation, immunoblot assays and human samples. (DOC) Acknowledgments The authors are grateful to Katt C. Mattos and Marcele Coelho for technical assistance. Author Contributions Performed the experiments: JCBF. Wrote the paper: JCBF PCB DMR. Contributed to study design: JCBF. Collaborated on experiments shown in Figure 4: BNB MG. Collaborated on experiments shown in Figure 2: PCB. Directed and designed the study: DMR. 5. Weekes J, Wheeler CH, Yan JX, Weil J, Eschenhagen T, et al. (1999) Bovine dilated cardiomyopathy: proteomic analysis of an animal model of human dilated cardiomyopathy. Electrophoresis 20: 898–906. 6. Fu HY, Minamino T, Tsukamoto O, Sawada T, Asai M, et al. (2008) Overexpression of endoplasmic reticulum-resident chaperone attenuates car- diomyocyte death induced by proteasome inhibition. Cardiovasc Res 79: 600–610. 7. Mearini G, Schlossarek S, Willis MS, Carrier L (2008) The ubiquitin- proteasome system in cardiac dysfunction. Biochim Biophys Acta 1782: 749–763. PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33175