the differentiated fiber [40,41,42]. Deregulation of RNF5 in this context is expected to interfere with the balance of these components most probably as a downstream component of this system, therefore altering the differentiation and the muscle maintenance programs. Of interest, RNF5 E3 ligase activity does not appear to be required for the muscular phenotype, as Tg mice that expresses RING mutant form of RNF5 exhibits similar phenotypes (data not shown). Consistent with this observation, a close homolog of RNF5, RNF185, was shown to control paxillin turn-over during Xenopus development independently of its E3 ligase activity [43]. Among possibilities to explain ubiquitin ligase independent effect of RNF5 is that RNF5 may impair muscle function by squelching or recruiting components of the ER machinery, by outcompeting its associated ligases (i.e. CHIP or gp78), or by acting as an E4 ligase on specific substrates after their ubiquitination by another E3 for which ligase activity may not always be required (i.e. p300 [44]). In conclusion, the characterization of the RNF5 transgenic and KO mouse models establishes an undisclosed link between impaired ER stress response and the early changes in degenerative myopathies, shown here for sIBM, while offering a novel model for studying the complex function of the ER in global muscle physiology. Methods Generation of RNF5 transgenic mice Mouse studies were performed under IACUC approvals from MSSM and BIMR. The mouse isoform of the RNF5 gene was cloned by PCR in frame with the HA tag into the pTRE2-HA vector using MluI and NheI restriction sites and sequenced. The linear fragment resulting from a HpaI-SapI digestion was then used for pronuclear injection. After microinjection into B6C3 (C57BL/ 66C3H) F2 hybrid eggs, the fertilized eggs were transferred into C57/BL6 female recipients and crossed with C57/BL6 males. Conditional RNF5 overexpression was achieved by crossing RNF5 STg animals with rtTA Tg mice, expressing the tetracycline- responsive Transcriptional Activator under control of the ubiquitous CMV-b-actin promoter, and the genotypes were verified by PCR reaction using the following primers (RNF5-forward: GTACCCATACGATGTTCCAGATTACGC; RNF5-reverse: CTGAGCAGCCAGAAAAAGAAAAAGATG; rtTA-forward: CGGGTCTACCATCGAGGGCCTGCT; rtTA-reverse: CCCGGGGAATCCCCGTCCCCCAAC); Both RNF5 and rtTA transgenic lines were kept as heterozygous and maintained as separate lines by crossing with WT C57/BL6 animals. MCK-tTA transgenic animals were obtained from Taconic Farms, as a repository for NIH deposited mouse strains and genotypes using the same primers as for rtTA animals. Immunoblot and immunohistochemistry analysis For expression analysis, frozen tissues were collected, flash frozen and pulverized using a mortar and pestle in liquid nitrogen. Proteins were extracted by resuspension in cold RIPA buffer containing anti- proteases and the extracts homogenized and clarified by high speed centrifugation. The protein concentration in the supernatant was determined by Bradford assay. RNF5 expression was analyzed either by straight immunoblot or after immunoprecipation using an affinity-purified RNF5 polyclonal antibody (1:2000 dilution). Expression for ER stress markers was assessed using rabbit GRP78 (Santa Cruz), rabbit GRP94 (Abcam) and mouse PDI (StressGene) antibodies and using GAPDH (Ambion) as a control. RNF5 in ER-Related Disorders For immunohistochemistry analysis, skeletal muscle tissues embedded in O.C.T. Compound (Sakura Tissue-Tek, product #4583) were pinned on cork pieces and snap-frozen in isopentane cooled in liquid nitrogen. Frozen tissues were stored at 280uC until cross-sections (8 mm thick) were prepared using a Leica CM3050S cryostat and stained. H&E stainings and Gomori-Trichrome were performed as previously described [45]. For immunostaining, sections were fixed in cold acetone for 5 min, permeabilized with 0.1% Triton X for 10 min and blocked with 1% glycine for 30 min. Immunostainings were performed using dystrophin antibody (Vector clone Dy8/ 6C5, diluted 1:20), CD45 (BD Pharmingen, clone 30-F11, diluted 1:200), Cd11b (BD Pharmingen, clone M170, diluted 1:100), embMHC (hybridoma bank F1.652, diluted 1:3), myogenin (hybridoma bank F5D, diluted 1:100), laminin a2 antibody (abcam 4H8-2,). RNF5 polyclonal antibody generated using full length bacterially produced RNF5. Rabbit’s serum was purified on RNF5 affinity column and used (dilution 1:100). For mouse monoclonal antibodies, the sections were incubated in biotinylated anti-mouse and avidin-conjugated fluorescein. (Mouse on Mouse Kit, Vector #FMK2201) For rat and rabbit antibodies, the sections were incubated with alexa fluor conjugated secondary antibodies and diluted 1:600 in Dako antibody diluent (#S3022) for 1 h at RT. Images were captured using an Olympus IX71 fluorescence microscope and the Slidebook version 4.0 software. Samples from human muscular disorders were obtained under IRB approval at the corresponding collaborating institutes in Italy and Japan. For the human sample processing, immunohistochemistry was performed on unfixed cryostat sections of diagnostic muscle biopsies from patients with the following diagnosis: s-IBM (10), DMD (2), BMD (2), normal muscle (3). After blocking, sections were incubated overnight at 4u with RNF5 antibody (1:100) and either monoclonal anti-phosphorylated tau SMI31 (1:5000, (Sternberger Monoclonals, Baltimore, MD, USA) or anti-beta amyloid protein (1:100, Biosource International, Camarillo, CA, USA, clone 6E10). Sections were incubated for 1 hr at RT with the appropriate Alexa fluor-conjugated secondary antibodies and Hoechst 33258 (Molecular Probes Inc.) staining was used to visualize cell nuclei. Slides were analyzed under a laser scanning confocal microscope SP5 (Leica, Germany) Phenotypic analysis of double transgenic and control animals A 2 mg/ml solution of doxycyclin supplemented with 5% sucrose was given to littermates animals between 12 wk and 24 wk of age in drinking water for 6 wk. Phenotypic alteration (body mass, weakness, blood withdrawal) were observed bi-weekly and the animals were sacrificed after 6 wk of treatment. Organs were individually weighed on a precision sale after trimming of the extra-tissues. Organs were then flash frozen in liquid nitrogen for expression analysis and frozen in OTC for immunohistochemical analysis. Electron Microscopy Glutaraldehyde-fixed muscle specimens were post-fixed in osmium tetroxide, and dehydrated in serial alcohol solutions and propylene oxide prior to embedding in araldite resin. Thick sections (1 mm) were stained with toluidine blue for light microscopy and ultrathin (60 nm) sections stained with uranyl acetate and lead citrate for electron microscopy. Morphometric analysis of skeletal muscle cross-sections Cross-sections of the triceps brachii, tibialis anterior, and vastus lateralis muscles from the double transgenic animals and their matching controls were immunostained with dystrophin and H&E PLoS ONE | www.plosone.org 12 February 2008 | Volume 3 | Issue 2 | e1609