Gastrocnemius and soleus muscles were powdered in liquid nitrogen before extraction. focal ML264 adhesions, such as vinculin and talin, are also elevated in mice (44). This increase in integrin and other components of adhesion complexes led us to suggest that the integrin and dystrophin complexes have overlapping roles in linking the extracellular matrix and cell cytoskeleton (36). Thus increasing the amount of integrin may functionally compensate for the absence of dystrophin in DMD patients. This hypothesis was tested and validated by showing that transgenic expression of 7 alleviates the development of severe muscular dystrophy in mice), extreme severe muscular dystrophies develop (1, 32, 58). These results confirm that the 71-integrin and dystrophin complexes are functionally complementary and important in maintaining skeletal muscle integrity. The beneficial effects of increasing integrin in polymerase (Stratagene, La Jolla, CA) using the following conditions: 95C for 4 min, followed by 35 cycles of 95C for 1 min, 65C for 30 s, 72C for 1 min, and a final extension at 72C for 10 min. The forward primer (5-ATGAATTCTCCCATGGCCAGGATTCCGAG-3) and reverse primer (5-TATCTAGAGCGAATTGGGTACACTTACCTG-3) were used. The amplified 7BX2 was cloned into pcDNA4/TO vector of the T-Rex system (Invitrogen, Carlsbad, CA), and the sequence of the final plasmid was verified. Animals Protocols for animal use were approved by the Institutional Animal Care and Use Committee, University of Illinois at Urbana-Champaign. The 7-integrin transgenic mice used in this study were previously described (7). Weights of 30 animals of each sex were measured weekly for up to 30 wk. Five-week-old female transgenic mice and their wild-type controls (SJ6/C57BL6) were used for microarray studies. Animals were euthanized by CO2 asphyxiation, and muscles were immediately dissected and snap frozen in liquid nitrogen. Cell culture and transfection C2C12 and L8E63 cells were cultured as previously described (9). Cells were transfected with linearized plasmids by using lipofectamine 2000 (Invitrogen) according to the manufacturers protocol. Stably transfected cells were selected with 7.5 g/ml blasticidin and 500 g/ml zeocin and were analyzed by immunofluorescence. Clones of the Tet7-C2C12 cells with the lowest basal level and the highest expression level of rat 7 inducible by 1 g/ml tetracycline were used for further studies. Antibodies Mouse monoclonal antibody O26 was used at a concentration of 10 g/ml to selectively detect rat 7-integrin by immunofluorescence (9). Polyclonal rabbit antibody against the Rabbit Polyclonal to NUMA1 7B cytoplasmic domain (CDB347) was used to recognize both mouse and rat 7-integrin (67). Monoclonal antibody O5 and CDB347 were used in Western blots to detect rat and total integrin 7, respectively (67). Rabbit polyclonal antibody against the cytoplasmic domain of the 1D integrin chain was generously provided by Dr. W. K. Song (Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, Korea). Monoclonal antibody against myosin heavy chain (MF20) was used to determine the fusion index. Rabbit anti-caspase-3 antibody (Cell Signaling Technology, Danvers, MA) was ML264 used to detect apoptotic myoblasts. Fluorophore and horseradish peroxidase-labeled secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). Skeletal muscle histology and fibers cross-section area Ten-micrometer sections of ML264 gastrocnemius muscle from 5-wk-old animals were frozen in liquid nitrogen-cooled isopentane (Sigma, St. Louis, MO), were cut by using a Leica CM1900 series cryostat (Nussloch, Germany), and were placed on microscope slides (Surgipath, Richmond, IL). Sections were fixed in 100% acetone at ?20C for 1 min, were rinsed in tap water for 10 min, and were stained with hematoxylin and eosin using standard histological procedures. Fiber cross-sectional areas were measured by using the advanced measurements component of Open-Lab software (Improvision, Lexington, MA). The.