to delineate the fibers, and the cross-section area of each fiber was quantified using Scion software (version 4.0.3.2; NIH). Evans Blue Dye Injection Evans Blue Dye (SIGMA) was diluted in PBS (10mg/ml) and filter- sterilized and the dye was injected through the tail vein at a concentration of 100ml per gram of body weight. Sixteen hours after injection, the mice were sacrificed and dissected muscles inspected for presence of blue dye in the muscle. Skeletal muscles were then fresh- frozen and cross-sections were further analyzed for presence of dye and counter-stained with dystrophin antibody as described above. Serum Creatine Kinase Assay 150 ml of blood was collected in heparin-treated tubes from periorbital bleeding and the serum fraction extracted by double centrifugation for 5 min at 5000 rpm. Creatine kinase levels were monitored using CPK-NAC kit (JAS Diagnostics #CPK1-15) and analyzed on a Roche Cobas Mira classic apparatus. Isolation of primary myoblast from muscle fibers One month old animals (RNF5 STg and rtTA RNF5 DTg) were sacrificed and tibialis anterior, gastrocnemius and soleus muscles were collected and rinsed in sterile PBS. Muscles were then digested in sterile filtered type 1 collagenase (0.2%) in DMEM using a shaking water bath for 2 hours at 37 degrees. Muscle fibers were then extracted by trituration after dilution of the digestion medium in one volume of DMEM. Single muscle fibers were then separated from debris by gently aspirating the fibers under a dissecting microscope and transferring them in a new dish containing DMEM plus 10% horse serum. This step was repeated 3 or 4 times in a row to separate intact fibers from the debris. 20 to 30 fibers were then plated in 60 mm dishes coated with 10% Matrigel and were incubated for 12–24 hours in DMEM plus 10% horse serum. Satellite cells were then allowed to proliferate for 3 days by changing the medium to DMEM plus 20% FBS, 10% horse serum, 0,5% chick embryo extract. Differentiation of myoblasts was subsequently achieved by changing the medium to 2% horse serum plus 0.5% chick embryo extract in DMEM. Doxycyclin (2 mg/ml) was added to the proliferation medium and the differentiation media to promote and maintain the expression of RNF5 transgene before and during the differentiation process. Immunocytochemistry Cells were washed in PBS and fixed using freshly prepared 3% paraformaldehyde in PBS (10 min at room temperature). The cells were then washed in PBS, followed by permeabilization in 0.1% Triton X-100 in PBS (pH 7.4) for 5min on ice and an additional three washes in PBS. Cells were then incubated in PBS supplemented with 3% bovine serum albumin for 30 min. The cells were incubated with antibodies (1 h at room temperature) in a humidity chamber. The cells were washed in PBS (36, 5 min each) before incubation with 100-ml of Alexa-488- and Alexa-568- conjugated anti-rabbit or anti-mouse immunoglobulin G (Molec- ular Probes) diluted (2 mg/ml) in PBS containing 3% BSA (60 min at room temperature in a light protected humidity chamber). The cells were rinsed three times in PBS and mounted on glass slides using Vectashield (Vector Laboratories). Primary antibodies were used as follows: RNF5 (1:200), sarcomeric alpha-actinin (Sigma, 1:5000), Pax7 (Hybridoma Bank, 1:10). Generation of RNF5 KO animals RNF5 targeting vector consisted of a 9kb 59 homologous region and 1.5 kb 39 homologous region of RNF5 genomic sequence which RNF5 in ER-Related Disorders was inserted into 59 of Neo gene cassette using PspOMI site. The Neo gene cassette was inserted to replace exon 1, 2 and 3 including the first ATG. Linearized targeting vector (10 mg) was transfected by electroporation into 129SvEv ES cells and screened for G418- resistant clones. Surviving colonies were expanded and PCR analysis was performed to identify clones that had undergone homologous recombination. Homologous recombination was confirmed by PCR using primer pairs, ZESA6 59-GCCAGCTGAAGGTGAGGGAC- TGGAC-39 and Neo1 59-TGCGAGGCCAGAGGCCACTTGT- GTAGC-39 The correctly targeted ES cell lines were microinjected into C57BL/6J blastocytes. The chimeric mice were generated and selected for a germline transmission of the disrupted RNF5 gene. Heterozygous mice were interbred to produce litters that included homozygous offspring. Additional analysis was carried out using DNA prepared from the MEFs that were obtained from these mice, further confirming the deletion of RNF5 gene. Chimeric mice with germline transmission were mated with C57BL/6J. The targeting vector and KO mice were generated by ITL, (Long Island NY). Backcross to C57BL/6J allowed to generate .90% strain. Genotyping were carried out by Southern blotting or PCR. Primers used for PCR were ZESA2 59-CCCTATGTCCTACAGGC- TCTG-39 and ZESA20 59-ACACGATGCTGAGGGGAGCTG- CAG-39 for the wild type allele, and ZESA6 and Neo1 for the targeted allele. Cardiotoxin treatment Four-month-old wild-type and RNF5 animals were anaesthetized with isoflurane, and 100 ml of 10 mM cardiotoxin (CalBiochem) in 1xPBS was injected into the right tibialis muscle. The animals were sacrificed at 3, 4, 6, 12 and 28 days after cardiotoxin injection. Tibialis muscles were harvested tendon-to- tendon and mounted in OCT or flash frozen in liquid nitrogen for histological or protein analysis. RNF5 silencing by siRNA The pCMS3-cherry vector was used to transfect short interfering RNA (siRNA) targeting RNF5 and its corresponding scramble control into Hela cells. Two 64-base complementary oligonucleotides (59GATCCCCAGCTGGGATCAGCAGAGA- GttcaagagaCTCTCTGCTGATCCCAGCT TTTTTGGAAA39 and 59AGCTTTTCCAAAAAAGCTGGGATCAGCAG AGA- GTCTCTTGAACTCTCTGCTGATCCCAGCTGGG-39) were synthesized to contain a RNF5 19-nucleotide sequence or its scramble counterpart ( 59-GATCCCC AGCTGGCATCAGCA- GGGAG ttcaagaga CTCCCTGCTGATGCCAGCT TTTTTG- GAAA 39 and 59AGCTTTTCCAAAAAAGCTGGCATCAG- CAGGGA GTCTCTTGAACTCCCTGCTGATGCCAGCTG- GG 39). The annealed product containing 59 and 39 overhangs compatible with BglII and HindIII restriction sites, respectively was then ligated into pCMS3-cherry digested with BglII and HindIII [46]. Supporting Information Figure S1 Specificity of RNF5 antibody. A. Specificity of RNF5 staining in cells where RNF5 expression has been downregulated by specific shRNA. HeLa cells were transfected with a plasmid expressing both the Cherry fluorescent marker and RNF5-specific shRNA or its scrambled version and analyzed for RNF5 expression by immunocytochemistry after 36 hours. B. Specificity of RNF5 antibody on muscle cross-sections. Triceps brachii cross- sections from RNF5 DTg or STg were stained with RNF5 antibody or a similar concentration of rabbit IgG. Arrowheads point areas of RNF5 staining. PLoS ONE | www.plosone.org 13 February 2008 | Volume 3 | Issue 2 | e1609