Journal: The Journal of Neuroscience
Article Title: Tenascin-R Is an Intrinsic Autocrine Factor for Oligodendrocyte Differentiation and Promotes Cell Adhesion by a SulfatideMediated Mechanism
Figure Lengend Snippet: Binding of TN-R 160 and TN-R 180 to polar GLs, as determined by solid phase ligand-binding assay. The fraction of brain polar GLs ( br. GLs , 2 μg/ml in PBS) or Sulf , GalC , galactosyl diglyceride ( GDG ), psychosine ( psy. ), GM1 , and sphingosine ( sphi. , all at 1 μg/ml in PBS) were coated into wells of microtiter plates, and the binding of biotinylated TN-R was determined after 2 hr of incubation at 37°C ( A ). B , Immobilized Sulf (at 0.5 μg/ml in 100% ethanol) was incubated with increasing amounts of TN-R 160 or TN-R 180 (2–50 μg/ml). Bound protein was detected by a subsequent incubation with polyclonal antibodies to TN-R (2 hr at 37°C), and antibody binding was visualized by using HRP-conjugated goat anti-rabbit IgG and ABTS (2,2-azino-di-[3-ethylbenzthiazoline sulfonate(6)]; Boehringer Mannheim). Background binding to BSA was subtracted from the values obtained for the binding to different GLs. The maximal absorbance at 405 nm for the binding of each TN-R isoform to Sulf was set as 1.0. Values represent the mean ± SD of three ( A ) or two ( B ) experiments performed in triplicate.
Article Snippet: Wells of microtiter plates (Falcon 3912; Becton Dickinson, Heidelberg, Germany) were coated with 100 μl/well of each GL (at a concentration of 0.5 or 1 μg/ml) for 1 hr at 37°C.
Techniques: Binding Assay, Ligand Binding Assay, Incubation