However, as the anti-GGA2 antibody gave less clear results compared with those of GGA1, we obtained only limited results from GGA2 staining of C2C12 cells

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However, as the anti-GGA2 antibody gave less clear results compared with those of GGA1, we obtained only limited results from GGA2 staining of C2C12 cells. GGA1 depletion Suxibuzone causes a defect in myotube formation of C2C12 cells To assess the physiological function of GGA1 in skeletal muscle mass differentiation, knockdown of was performed in C2C12 cells. Multinuclear myotube (arrow) showed IR staining whereas little or no staining was seen in the mononuclear myoblasts (arrow heads).(TIF) pone.0207533.s003.tif (4.5M) GUID:?5DF4AB29-8B04-4C72-A96E-DAAEC2768E4C S4 Fig: proximity biotin labeling of IR by GGA1-BirA. To perform the proximal biotinylation, the ORF of GGA1was subcloned into pcDNA3.1 MCS-BirA(R118G), which was a gift from Dr. Kyle Roux (Addgene plasmid #336047)[35]. (A) pcDNA3.1 MCS-BirA(R118G) (BirA-HA) and pcDNA3.1-GGA1-BirA(R118G) (GGA1-BirA-HA) were transfected into C2C12 cells and intracellular localization of BirA-HA (a) and GGA1-BirA-HA (b) was examined by immunofluorescent microscopy with anti-HA antibody. The GGA1-BirA was localized at the perinuclear Gogi area (arrows) and at the peripheral puncta (arrow heads), whereas BirA alone did not localized at any intracellular compartments. (B) C2C12 cells were subjected to differentiation for 4 days and transfection of BirA-HA and GGA1-BirA-HA expression constructs was performed. Twenty-four hours after transfection, cells were incubated with 50 mM biotin for 6 hours and lysed. The biotinylated proteins were captured by streptavidin-sepharose (Wako). The input total lysate (5%) and SMAD9 the biotinylated proteins were subjected to immunoblotting with anti-IR antibody. The result indicated that GGA1-BirA-HA successfully biotinylated IR mRNA expression was upregulated during myogenesis, and depletion prevented the formation of large multi-nucleated myotubes. Moreover, inhibition of lysosomal proteases in knockdown myoblasts increased the amount of insulin receptor, suggesting that GGA1 is usually involved in the cell surface expression and sorting of the insulin receptor. These results suggested that GGA1 plays a significant role in the formation and maturation of myotubes by targeting the insulin receptor to the cell surface to establish functionally mature myofibers. Introduction Skeletal muscle tissue has essential functions within the body, such as movement, metabolism, glycopexis, and thermoregulation [1]. During muscle mass development, or muscle mass repair after damage, muscle mass satellite cells have crucial functions in the generation of muscle mass fibers. First, quiescent satellite cells are activated to become myoblasts and their number increase. Second, the differentiated myoblasts migrate into the damaged areas within the muscle mass. Third, multi-nucleated myotubes are created through myoblast-to-myoblast or myoblast-to-myotube cell fusion [1]. The formation, maintenance, and growth of healthy skeletal muscle tissue are dependent upon these elementary actions. During myogenic differentiation, myoblast cells undergo drastic changes in cell shape as a result of cell-to-cell fusion, becoming large, multi-nucleated myotubes that are the functional precursors of skeletal muscle mass cells. In the course of this differentiation, the secretion of several growth factors [2,3] and the cell surface expression of the fusion machinery are essential for proper muscle mass generation [4,5]. Therefore, the intracellular protein trafficking system is usually thought to play a significant role in the stage-specific protein secretion and sorting of several plasma membrane proteins required for myogenesis. Protein sorting at post-Golgi organelles requires the formation of carrier vesicles, such as clathrin-coated vesicles. A group of proteins termed clathrin adaptors is usually involved in the recognition of the cargo molecules and the physical formation of the membrane-bound clathrin-coated vesicles from your [7], it is also believed that each GGA has its specific interactors. For example, the GAT domains of GGA1 and GGA3 have higher affinity for ubiquitin compared with that of GGA2 [11]. Recently, Uemura et al. Suxibuzone showed that p56, an accessory protein of GGAs, is usually localized at the TGN in a GGA1-dependent manner [12,13]. In addition, while single knock-out (KO) of or caused no obvious phenotypes in mice, Suxibuzone the double KO or single KO mice were embryonic lethal [9,10]. These results strongly suggested that each GGA has specific physiological functions in C2C12 cells C2C12 cells were purchased from ATCC (#CRL-1772) and cultured in growth medium made up of Dulbeccos altered Eagles medium (DMEM) (Wako, Osaka, Japan) with 15% fetal bovine serum and 1% penicillin-streptomycin (growth medium). Muscle mass differentiation of C2C12 cells was induced by changing Suxibuzone the medium to DMEM supplemented with 2% fetal bovine serum and 1% penicillin-streptomycin (differentiation medium) for 4 days. To knock down gene expression, C2C12 cells were transfected with 20 nM of gene-specific small interfering RNA (siRNA, ON-TARGETplus SMART pool, Dharmacon) using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA, USA). For control experiments, cells were transfected with non-targeted siRNA (Sigma, St Louis, MO, USA). To measure insulin-dependent glucose uptake, cells were shifted to a serum-free medium for 13 h and treated with Krebs-Ringer-Phosphate-HEPES buffer made up of 2% (w/v) bovine serum albumin.