Figure 6.

Phosphorylation of A7 increases its binding to SNAP23. A. Recombinant A7 was incubated for 2 h at 30° C without or with PKC or ATP and MgCl₂ and binding to Sepharose4B-bound GST-SNAP23 fusion protein was evaluated in presence of 100 μM Ca²⁺. The binding was increased for A7 incubated with PKC and ATP. B. In separate experiments, A7 was phosphorylated for 2 h without or with PKC and binding to Sepharose4B-bound GST-SNAP23 fusion protein was measured following two hours incubation at 4° C in presence of indicated Ca²⁺ concentrations. The binding of A7 was evaluated by western blot analysis. C. Left Panel. Densitometry analysis of western blots from three experiments is shown as mean ± SE of protein (a.u., arbitrary units) on the blot. * P < 0.05 for non-phosphorylated A7 in comparison to zero Ca²⁺, ** P < 0.05 for phosphorylated in comparison to non-phosphorylated A7 at each Ca²⁺ concentration. Right Panel. Densitometry analysis shows equivalent levels of A7 in the assay mixture in the absence or presence of PKC (last two lanes in B).