Cells treated with the Nogo-A peptide showed significant changes in expression, which occurred in a short period of time, resulting in a significant decrease in NFL expression

Cells treated with the Nogo-A peptide showed significant changes in expression, which occurred in a short period of time, resulting in a significant decrease in NFL expression.27 In addition, the messenger levels obtained after Nogo-A antibody treatments at different concentrations showed a restoration of gene expression levels in a dose-dependent manner (Figure 5(b)). of function below the initial site of the injury, permanently removing the patients ability to perform basic daily activities. The loss of function occurs by disrupting signal transduction mechanisms of both ascending and descending neuronal tracts in the gray matter of the spinal cord. After the primary compression or contusion injury, a secondary injury cascade occurs leading to the formation of a glial scar, an increase in cell death at the injury site, and an upregulation of inhibitory factors that prevent functional recovery.2,3 Although there is no cure for SCI yet, a better understanding of the molecular and cellular mechanisms post-injury, with advances in the fields of biomaterials and tissue engineering, Rabbit polyclonal to Transmembrane protein 132B has led to important new discoveries.4 It is well established that neurons of the central nervous system (CNS) can extend neurites after injury when offered a permissive environment.5,6 The endogenous inhibition of this system has been recognized by cell surface receptor and myelin interactions.7 Since 2005, the transmembrane protein Nogo-A has been recognized as one of the primary contributors for neurite outgrowth inhibition.8,9 This finding has been further supported by the application of specific antibodies against Nogo-A using in vitro as well as in vivo models.10 These findings demonstrated that a Nogo-A antibody functions by blocking the interaction of Nogo-A with the Nogo receptor and co-receptors p75, TROY, and LINGO-1 located on neurons from the CNS.11C13 Various anti-Nogo-A antibodies have been engineered and reported in the literature.14,15 One of the most commonly studied Nogo-A antibodies is the mouse monoclonal antibody 11c7, which was raised against a peptide corresponding to aminoacids 623C640 of the rat Nogo-A protein sequence,8 which are within the central inhibitory domain.16 The in vitro effects of the 11c7 antibody have been previously shown Cinchophen by Western blot, as well as indirect immunoassays utilizing variety of cell-based assays.17,18 While several cellular and non-cellular assays have been established to quantify the effects of Nogo-A activation,8,19 the development of a reliable approach to characterize the antibody is important for SCI recovery. Whereas the delivery of anti-Nogo-A has been of great interest for neuroregeneration, our long-term goal is to develop a localized delivery Cinchophen strategy for this antibody with the goal of facilitating neuroplasticity at the injury site.20C23 The main objective herein is to report for the first time a systematic approach to evaluate the anti-inhibitory effects of the Nogo-A antibody 11c7 on the nerve growth factor (NGF)-induced neuronal-like differentiation of the pheochromocytoma cell line, PC-12. By following this approach, a new in vitro cell-based assay to facilitate quantification of an anti-Nogo-A antibody has been demonstrated. Description of methods Materials The mouse monoclonal antibody 11c7 against Nogo-A was generously provided by Novartis Pharma AG (Basel, Switzerland). Dulbeccos phosphate buffered saline (PBS; pH 7.4) was purchased from Invitrogen (GIBCO Invitrogen Laboratories, NY, USA). All buffers were prepared using water distilled and deionized Cinchophen using a Millipore Milli-Q UF Plus at 18?M resistance (Millipore, Bedford, MA, USA). Cell culture The rat adrenal pheochromocytoma cell line, PC-12, was purchased from the American-type culture collection (CRL-1721, ATCC, Rockville, MD, USA), cultured in RPMI 1640 with UltraGlutammine media (LONZA, Basel, Switzerland), and supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS), and 1% penicillin/streptomycin.