Antibodies to the N-terminal and Rho-GEF domains labeled stretched samples at the level of the M-band, and also at the level of the I-band (Fig 3A,D,G,J; compare with labeling by antibodies to Z-disk epitopes of titin, Fig 3B,E, and -actinin, Fig 3H,K)

Antibodies to the N-terminal and Rho-GEF domains labeled stretched samples at the level of the M-band, and also at the level of the I-band (Fig 3A,D,G,J; compare with labeling by antibodies to Z-disk epitopes of titin, Fig 3B,E, and -actinin, Fig 3H,K). M-band. Obscurin B (~900kDa) which has the N-terminal, Rho-GEF, and the C-terminal kinase-like domains, CXCR7 localizes at the level of the A/I junction. Faldaprevir Additional isoforms, which lack one or more of these epitopes, are present at the Z-disk and Z/I junction. gene may have unique roles in myofibrillogenesis. Understanding the subcellular distribution of obscurin is usually complicated by the presence of multiple alternatively spliced forms. Obscurin is present in skeletal muscle in an ?800 kDa form, now termed obscurin A [4], characterized by a C-terminal non-modular region that binds a small form of ankyrin in the network sarcoplasmic reticulum [7,9]. An additional variant, with a larger molecular mass than obscurin A arises from the replacement, by alternative mRNA splicing, of the non-modular carboxy terminus with two serine/threonine kinase-like domains. Originally identified as the giant kinase isoform of obscurin [2,10,11] it has been more recently termed obscurin B (Fig 1) [4]. Obscurin is also expressed as several smaller alternatively spliced variants [1,2,4,5,12]. Several alternatively spliced products have also been identified for obscurins homologue in [13]. Open in a separate window Physique 1 Obscurin isoformsA.Antibodies were prepared to distinct regions of obscurin: the N-terminal Ig domain name, and the Rho-GEF domain name, the non-modular C-terminus of obscurin A and to Ob68 adjacent to the first kinase-like domain name (SKII) in the giant kinase isoform, obscurin B. We used antibodies to different regions of obscurin, the N-terminus, the C-terminus, the Rho-GEF domain name, and the Ig domain name N-terminal to the first kinase-like domain name (Ob68) to characterize the subcellular location of some of the alternatively spliced forms Faldaprevir of obscurin in skeletal muscle at resting length and after stretch (Fig 1). We localized obscurin A and obscurin B to the M-bands. In addition, we found obscurin B near the A/I junction, and localized distinct obscurin domains, or combinations thereof, likely to be indicative of other, alternatively spliced forms, to the Z-disk and at the Z/I junction. The presence of two and possibly more different forms of obscurin at distinct sarcomeric locations suggests that these proteins play several distinct roles in organizing and stabilizing the contractile apparatus and nearby structures. MATERIALS AND METHODS Antibodies We used the reverse transcriptase polymerase chain reaction (rtPCR) to amplify the coding region of the Rho-GEF domain name of obscurin from rat skeletal muscle poly A+RNA (Clontech, Palo Alto, CA) using the Superscript First Strand Synthesis System (Invitrogen, Carlsbad, CA). The primers were: reverse transcriptase, GCCACAGATCTGCTTCACCCA; forward, AGTGAATTCGTCATCCAGGAGTTGCTGAGTTC; reverse, ATCGGATCCCTAGCGCTGTGGCAGGGCAGA. This cDNA was cloned into the EcoRI/XhoI sites of pGEX-4T-1. Synthesis of the glutathione-(EDL) muscles, removed from adult rats with proximal and distal tendons intact, were placed in Krebs solution equilibrated with 95% O 2 , and 5% CO 2 , pH 7.2. Silk threads (#4) were tied around the tendons and muscles were extended to L o, the resting tension Faldaprevir length from the proximal to the distal myotendinous junction [ 18 , 19 ] , or to distances ranging from 1.3-1.7Lo. (Only smaller bundles of fibers could be stretched to 1 1.7Lo, due to the passive resistance of the connective tissue in intact muscle). The muscles were fixed immediately in 2% paraformaldehyde in PBS for 20 minutes, snap frozen, and cryosectioned [9]. Fluorescent Immunolabeling and Confocal Microscopy Frozen longitudinal sections were incubated in PBS made up of 3% BSA and 5% non-immune goat serum (PBS/BSA/NGS) followed by primary antibodies in PBS/BSA/NGS overnight at 4C [9]. Samples were washed, incubated with species-specific secondary antibodies in PBS/BSA/NGS, mounted in Vectashield, and observed with a Zeiss 410 confocal laser scanning microscope (Carl Zeiss, Inc., Tarrytown, NY). RESULTS Localization of obscurin epitopes in skeletal muscle at resting sarcomere lengths We used immunofluorescence to identify sites in resting muscle fibers labeled by antibodies to different epitopes of obscurins A and B. The specificity of the C-terminal and SKII antibodies have been previously described [5,9,16]. In addition, we performed immunodepletion experiments demonstrating that immunoreactivity was eliminated by preadsorbtion with the immunogen (Fig 2A, D, G, J, inset). Antibodies to the N-terminus primarily recognize structures at the level of the M-band, with additional but faint labeling near the Z-disk (Fig 2A), as determined by double immunofluorescence with antibodies to epitopes of titin found at the Z-disk (Fig 2B, C). Double immunofluorescence experiments with antibodies to -actinin (Fig 2E, F) indicate that this Rho-GEF domain name of obscurin is usually primarily found in structures at the level of the M-band (Fig 2D). As previously reported, antibodies to the C-terminal region of obscurin A, and to the Ob68-serine-threonine kinase region of obscurin.