Figure 5.
Synaptotagmin I labeling of cerebellar cortex and β4a-A interaction with 6His–C2AC2B in pull-down assays. A, Immunolabeling of mouse cerebellar cortex with anti-synaptotagmin antibody (scale bar, 20 μm). ML, Molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. B, CD spectrum of recombinant purified 6His–C2AC2B indicative of proper folding into a β-sheet structure. C, Pull-down assays analyzed by SDS-PAGE, using Coomassie blue-stained 5–15% polyacrylamide gels. Equal amounts (50 nmol each) of β4a-A and β4b-A (lanes 1, 2) were incubated with 6His–C2AC2B–Ni2+-NTA beads and washed until neither β4a-A or β4b-A appeared in the flow through (lanes 3, 4). Bound 6His–C2AC2B and any associated protein were eluted with 1 m imidazole. Lanes 5 and 6 show that β4a-A, but not β4b-A, associates with 6His–C2AC2B in the absence of added Ca2+. The interaction of β4a-A with 6His–C2AC2B does not occur when 10 mm Ca2+ is added before the wash step (PreW, lane 7). Addition of 10 mm Ca2+ to the assay after the wash step but before the imidazole elution competes off β4a-A (PostW, lane 8). Subsequent addition of 1 m imidazole elutes off 6His–C2AC2B (Imid, lane 9).