1 Introduction [1] 2+ [2] N F A T [3,4] [5] [6-9] [10,11] [12-16] [17] [18] 3 2+ 2+ 2+ i 2+ 2+ [3] 2+ [19-21] [22] 2+ 2+ [23,24] [25] [26] 2+ [23,27] 2+ 2+ i 2+ i 2 Materials and methods 2.1 Recombinant proteins [26] 2.2 Small Scale GST protein binding assays 2 2+ 2+ 2 2+ 2+ 2.3 Large scale GST protein binding assays [26] 2+ 2+ 2+ 2+ 2.4 Sulfo-SBED cross-linking experiments The manufacturers protocol provided by Pierce (Rockford, IL, USA) was followed with minor modifications. All steps, except those indicated, were carried out in the dark. Bait recombinant protein of interest (∼5 mg) was dialysed against binding buffer overnight at 4 °C. Immediately before use, the contents of one tube of No-Weigh Sulfo-SBED (Pierce) was dissolved in 22 μl of DMSO, this was then added to 1 ml of dialysed protein. Sulfo-SBED/protein mixes were incubated at room temperature for 30 minutes, centrifuged briefly to remove any precipitated, hydrolysed, Sulfo-SBED, and dialysed overnight against binding buffer. Bovine brain cytosol (500 μl, ∼4 mg protein) dialysed against binding buffer was added to 500μl of the Sulfo-SBED/protein mix. This mixture was incubated on a rotator at room temperature for 1 hour. After this step, samples were exposed to long-wave UV illumination (365 nm) at a distance of ∼5 cm from source for 15 minutes. During this step an appropriate volume of Neutravidin beads (Pierce) were washed extensively in binding buffer, 100 μl added to the bait/cytosol mixture and samples incubated on a rotator for 1 hour at room temperature. Bait/cytosol mixes were then centrifuged at 13,000 rpm for 1 minute, supernatants removed and beads washed with 1 ml of binding buffer. This step was repeated 6 times. Disulfide bond reduction was then achieved by incubating with 1 ml of 50 mM DTT for 1 hour at room temperature. Beads were subsequently centrifuged and washed extensively with binding buffer. After the final wash, 100 μl of SDS dissociation buffer was added to the beads and samples boiled for 10 minutes. Samples were separated on SDS PAGE (12.5% gel) and western blotted with a monoclonal anti-calcineurin antibody (1:1000, Sigma) followed by detection with anti-mouse-HRP (1:400, Sigma) and ECL reagents. 2.5 HeLa cell cultures and transfections 2 2 2.6 Ionomycin induced NFAT translocation 2 2 2 4 2.7 Confocal microscopy For confocal laser scanning microscopy, transfected cells were examined with either a Leica TCS-SP-MP microscope or a Leica TCS-SP2-AOBS microscope (Leica Microsystems, Heidelberg, Germany) using a 22 μm pinhole and a 63× water immersion objective with a 1.2 numerical aperture. EGFP was imaged using excitation at 488 nm and light collection at 500-550 nm. 3 Results 3.1 Direct binding of calcineurin to NCS proteins 2+ Fig. 1 Fig. 1 Fig. 1 [28] [26] 2+ 2+ 2+ Fig. 1 C F 2+ Fig. 1 H S 2+ Fig. 1 2+ [29] Fig. 1 Fig. 1 Fig. 1 Fig. 1 Fig. 1 2+ Fig. 1 Fig. 1 2+ 3.2 2+ [30] 2+ [30,31] [32-35] [25] [32] E120Q 2+ a [32-36] G2A [37,38] . Fig. 2 2+ 2+ i Fig. 2 2+ i Fig. 2 Fig. 2 2+ Fig. 2 Fig. 2 2+ Fig. 2 Fig. 2 Fig. 2 [39] Figs. 2 G2A E120Q Fig. 2 3.3 E120Q G2A 2+ 2+ i 2+ i Fig. 3 2+ 2+ 2+ 2+ 2+ 2+ [40] 2+ [41-43] Figs. 2 and 3 E120Q G2A 2+ . E120Q E120Q Fig. 3 2+ 2+ 2+ 2+ 4 Discussion 2+ 2+ 2+ [2] 2+ [23] [25] bone fide [25] [3] [25] 2+ [44] 2+ et al. [45] d [46] [25] [25] 2+ i 2+ i [26] [47] E120Q G2A [32] [32-35] G2A 2+ 2+ [40,48,49] 2+ [50] [34,51,52] [35]