Figure 7.

Physical interactions of the (α2)δ1 peptide with σ1Rs, HINT1 proteins, and CaM. (A) Calcium-dependent binding of σ1Rs to (α2)δ1 peptides. Recombinant (α2)δ1 peptides covalently attached to NHS-activated Sepharose^® were incubated with σ1Rs (100 nM) in the presence of increasing amounts of CaCl₂. The pellets obtained were washed, solubilized in 2× Laemmli buffer containing β-mercaptoethanol, and resolved by SDS-PAGE. The presence of σ1R was analyzed in Western blots (WBs). The prey protein did not bind to NHS-Sepharose (O + σ1R, negative control). In another set of assays, (α2)δ1 peptides were incubated with increasing concentrations of σ1Rs in the presence of CaCl₂ (2.5 mM). (B–D) Competition assays between σ1R, HINT1, and CaM for their binding to (α2)δ1 peptides. CaM (100 nM) was incubated with agarose-(α2)δ1 for 30 min at RT in 300 μL of 50 mM Tris-HCl, [pH 7.5], 0.2% CHAPS, CaCl₂ (2.5 mM). After removal of the unbound CaM, increasing concentrations of σ1Rs were added. The (α2)δ1-bound proteins were detached, resolved by SDS-PAGE chromatography, and analyzed in Western blots (see Section 2). The assays were repeated at least twice, producing comparable results. This protocol was also used to assess competition between HINT1/σ1R and CaM/HINT1 in their binding to (α2)δ1 peptides. O and Θ represents plain agarose and NHS-Sepharose^®, respectively.