Fig. 1.

NCS proteins interact with calcineurin in vitro. (a) Upper Panel, Anti-calcineurin western blot of samples from an in vitro binding assay examining interaction of recombinant CN (1 μM) with various GST-tagged NCS proteins (all at 1 μM) and to calmodulin sepharose (1 μM) in the presence of buffer containing 1 μM free Ca²⁺. CN was associated directly with calmodulin sepharose and to a lesser extent with GST-NCS-1, GST-Hippocalcin, GST-Neurocalcin-δ and GST-CaBP1 but not with GST-KChIP1. (a) Lower panel, coomassie blue stained gel highlighting equal loading of GST fusion proteins input into the binding assay. (b) Anti-calcineurin western blot of samples from an in vitro binding assay examining the ability of CN derived from bovine brain cytosolic extract to associate with GST control and GST-NCS proteins in both the absence (High Salt or HS) and presence (Calcium Free or CF) of 1 μM free Ca²⁺. CN was detectable bound to both GST-NCS-1 and GST-CaBP1 only in the presence of free Ca²⁺ and no CN was present in HS samples or GST control protein samples either in the presence or absence of Ca²⁺. The amount of CN in the bovine brain cytosolic extract was shown in a positive control sample of total brain cytosolic lysate resolved on the same gel (Bovine brain cytosol). (c) Anti-calcineurin western blot of samples obtained from a Sulfo-SBED cross-linking experiment (see Materials and Methods for details). CN was detectable in samples from a GST-neurocalcin-δ affinity column treated with the biotin transfer reagent ((+) Sulfo) but not in its absence ((-) Sulfo). Input bovine brain cytosolic extract was resolved on the same gel (Bovine brain cytosol). (d) Anti-calcineurin western blot on samples from in vitro binding experiments involving incubation of bovine brain cytosolic protein extract with GST control protein, calmodulin sepharose and GST-NCS-1 (all at 1 μM) in the absence (-) or presence (+) of 1 μM free Ca²⁺. Total bovine brain cytosolic protein extract was separated on the same gel as a positive control for CN.