1993; Wayne et al. website that transmits signals from nerve terminals to the muscle mass dietary fiber (Tintignac et al. 2015). The myotendinous junction (MTJ) is the website that transmits causes from muscle mass to tendon (Tidball 1991). The extrasynaptic, or extrajunctional, website links the sarcolemma to the ECM to transmit pressure during muscle mass Levobunolol hydrochloride contraction. These unique domains consist of discrete units of membrane molecules required for their different functions. Within the extrasynaptic sarcolemma, a major adhesion complex is the dystrophinCglycoprotein complex (DGC). The DGC links the intracellular actin cytoskeleton to the ECM via the laminin receptor dystroglycan and the intracellular adaptor protein dystrophin (Ervasti and Campbell 1993). A homologous complex, the utrophinCglycoprotein complex (UGC), which localizes to the NMJ and MTJ domains, also includes dystroglycan but uses utrophin rather than dystrophin to link the ECM to the cytoskeleton (Clerk et al. 1993; Wayne et al. 1995). Dystroglycan is definitely a heterodimer of soluble alpha-dystroglycan (-DG) and membrane-bound beta-dystroglycan (-DG). -DG offers N- and C-terminal globular domains linked by an extended mucin website. The C-terminal globular website links -DG to -DG, while the highly glycosylated mucin website bears unique Rabbit Polyclonal to MAP3K7 (phospho-Thr187) agglutinin (DBA), a flower lectin that recognizes terminal agglutinin (VVA) and agglutinin (WFA) have been used to differentiate the NMJ from extrasynaptic sarcolemma (McDearmon et al. 2001; Nguyen et al. 2002; Marshall et al. 2012). WFA specifically has been used to differentiate between complexes associated with NMJ vs. the extrasynaptic sarcolemma. WFA preferentially binds to -DG in the UGC, while the sialic acid and mice. As previously described, we observed distributing of WFA reactivity throughout the sarcolemma in muscle mass from mice, which correlated with utrophin staining. However, VVA, SBA and DBA staining remained highly specific for the NMJ in muscle mass from mice. Interestingly, we mentioned similar differences between the four GalNAc-specific lectins when muscle mass from 7-integrin-null (7?/?) mice was stained. While VVA, SBA Levobunolol hydrochloride and DBA staining remained highly NMJ specific, WFA staining spread extrasynaptically and correlated with utrophin staining. As WFA staining redistributed concomitantly with utrophin staining, we next asked if manifestation of utrophin was necessary for staining of WFA Levobunolol hydrochloride or the additional GalNAc-specific lectins. Remarkably, we observed no loss of reactivity for any of the four lectins with muscle mass from utrophin-null (Utr?/?) mice compared to crazy type, Levobunolol hydrochloride indicating that utrophin manifestation is not required for binding of WFA, VVA, SBA or DBA (Number ?(Figure11). Open in a separate windows Fig. 1. The DGC, UGC and 7-integrin are not requisite for binding of GalNAc-specific lectins. Transverse 8-m serial cryosections of murine quadriceps muscle tissue from 12-week-old wild-type, on-line. Once we observed some qualitative variations in patterns of reactivity of WFA, VVA, SBA and DBA with muscle mass from wild-type and mice, we developed an in vitro lectin binding assay to quantify variations in binding among the lectins. We used a panel of 11 lectins (Table ?(TableI)I) to analyze glycosylation of C2C12 murine myoblasts and differentiated myotubes. Fixed cells were incubated with biotinylated lectins and bound lectins were recognized colorimetrically. In addition to the GalNAc-specific lectins WFA, VVA, SBA and DBA, the panel included lectins specific for major classes of glycan constructions found on glycoproteins.?WGA, agglutinin (SNA) and agglutinin-II (MAA-II) were included for specificity for terminal sialic acid residues (Greenaway and LeVine 1973;.