Article previously (7) and were bred and maintained on a C57BL/6 back- ground at Universite Joseph Fourier. Animal care and proced- ures were approved and performed in accordance with the stand- ards set forth by the Institutional Ethics Committee, the National Research Council Guide for the care and use of laboratory ani- mals, the National Institutes of Health, and the Animal Care Use and Review Committee of the University of Iowa. Preparation of Antibodies and Microsomes. mAbs against CSQ (clone VIIID12, Affinity BioReagents) and Cav-3 (BD Transduction Laboratories) were used as described in the com- pany datasheet. The sheep anti-DHPR R1 subunit was obtained from Upstate Biotechnology. Polyclonal antibodies against the RyR, the common N-terminal end of tradins, Trisk 95, and Trisk 51 were described previously (2, 3, 11). Guinea pig anti-Ca2þ- ATPase was a gift from A. M. Lompre (12). Crude microsomes were prepared from 1-month-old mouse gastrocnemius muscle as previously described (13). Viruses. The viruses were engineered and produced by the Gene Vector Production Network, at Genethon III (Evry, France). Three type 5 adenoviruses were used in this study, a control virus (AdV-DsRed) with the cDNA of the red fluorescent protein (DsRed), AdV-Trisk 95, an adenovirus with the full-length seq- uence of rat skeletal muscle T95 (EMBL AJ243304, 687 amino acids), and AdV-Trisk 51, an adenovirus with the full-length sequence of rat skeletal muscle T51 (EMBL AJ243303, 461 amino acids). All the transgenes were under the control of a CMV promoter. In Vivo Infection. Injections of adenovirus into 3-4-day-old wild-type pups were performed as previously described (14), with some modification: the hamstring, quadriceps, calf, and tibialis anterior muscles of one leg were each injected percutaneously with 1010 pfu diluted in 10 μL of saline; the same muscles of the contralateral leg were each injected with an equal volume of saline. Pups were reintroduced to the mother and kept in quarantine for 5 days. All pups survived after injections. Muscles were harvested 30 days after injection, snap-frozen in liquid nitrogen-cooled isopentane, and stored at -80 C until they were used. Cell Culture and Cell Infection. Rat myogenic L6 cells (clone C5) or COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin. L6 cells were infec- ted at a multiplicity of infection (MOI) of 200 with the adenovir- uses encoding T95 (AdV-Trisk 95) or encoding T51 (AdV-Trisk 51). The cells were collected 48 h after infection. Plasmids and Transfection. The transmembrane domain of human RyR1 (GenBank accession number J05200) corresponding to amino acids 4457-5038 was cloned in pEGFP-C1 (Clontech), to produce a 93 kDa fusion protein corresponding to the GFP fused with the channel part of RyR1. Human caveolin 3 (Gen- Bank accession number AF036365) was cloned in pCDNA3.1 (Invitrogen). COS-7 cells were transfected with the two plasmids (2.5 μg of each plasmid for 8 105 cells) using ExGen 500 (Euro- medex), and the cells were collected 24 h after transfection. Immunoprecipitation. Five hundred micrograms of micro- somes from rat skeletal muscle or 300 μg of cells was solubilized at 2 mg/mL in the presence of 1.6% CHAPS, 0.9 M NaCl, 0.1% phospholipids, 100 μM CaCl2, 50 μM EGTA, 20 mM Pipes (pH 7.1), and protease inhibitors (1 mM diisopropyl fluoropho- sphates and 100 μM phenylmethanesulfonyl fluoride), and im- munoprecipitation was performed with antibodies against rat Trisk 95, rat Trisk 51, or nonimmune serum as described previ- ously (2), using 10 mg of protein A immobilized on Sepharose 4B. Biochemistry, Vol. 49, No. 29, 2010 6131 All the immunoprecipitated proteins were then analyzed by Western blotting. Western Blot. The presence of RyR, DHPR, Trisk 95, Trisk 51, calsequestrin, Ca2þ-ATPase SERCA, or caveolin 3 in dif- ferent samples was assayed by Western blotting, using a chemi- luminescent reagent (Western lightning Chemiluminescence re- agent plus, PerkinElmer Life Science). After electrophoretic separation on a 5-15% acrylamide gel, the proteins were electro- transferred to Immobilon P (Millipore) as previously described (3). The secondary antibodies were labeled with horseradish per- oxidase (Jackson ImmunoResearch Laboratories). Quantitative analysis was performed using a Chemidoc XRS and Quantity One (Bio-Rad), and the amount of each protein was normalized to the amount in AdV-DsRed-infected muscles, after loading correction by Coomassie blue staining of myosin, as described previously (15). Immunofluorescence and Histological Analyses. Seven micrometer cryosections were prepared and analyzed by immu- nofluorescence or H&E staining as previously described (16). For immunofluorescent staining, sections were washed with phos- phate-buffered saline (PBS), blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature, and incu- bated with a primary antibody in a PBS/1% BSA mixture over- night at 4 C. After incubation for 1 h at room temperature with an Alexa fluor 488-conjugated (Invitrogen) or Cy3-conjugated secondary antibody in a PBS/1% BSA mixture, the samples were mounted with PermaFluor (Beckman Coulter). Sections were observed under an MRC-600 laser scanning confocal microscope (Bio-Rad). Digitized images were captured under identical con- ditions. Images of H&E staining were photographed using a Leica DM RXA microscope equipped with an Olympus DP70 digital camera. RESULTS Effect of Trisk 95 and Trisk 51 Overexpression on the Expression of the CRC Components. Trisk 95 and Trisk 51, the two major skeletal muscle triadin isoforms, were overex- pressed in vivo using injection of adenovirus into newborn mice. As a control, the muscles were injected with an adenovirus en- coding DsRed. To determine the effect of Trisk 95 or Trisk 51 overexpression on the expression of their calcium release com- plex’s partners, immunoblot analysis of crude muscle extracts was conducted 30 days after gene transfer (Figure 1A,B). Protein expression levels were normalized to the amounts measured in control muscles infected with AdV-DsRed (Figure 1A, lane 3). Both Trisk 95 and Trisk 51 were overexpressed by 60% (Figure 1A, lanes 1 and 2, and Figure 1B), but the expression levels of the RyR, the R1 subunit of DHPR, CSQ, and SERCA were not changed (Figure 1B). This experiment was conducted on muscles from different animals, with similar results (no modification of the expression level of the proteins assayed, except for that of the overexpressed triadin). Nevertheless, the level of overexpression depends on the infection level, which is not identical among all the animals. Using Western blot on muscle homogenate, the infected fibers are mixed with noninfected fibers, which constitute the majority of the fibers. To discriminate between infected and noninfected fibers and to evaluate more precisely the modification induced on the other proteins, we chose to analyze the expression of the CRC components in Trisk 95- and Trisk 51-overexpressing muscles by immunofluorescence. While the stainings for RyR, DHPR, and CSQ were unchanged in Trisk 95-infected fibers,