Figure 4.

CaBP1 binds directly to the InsP₃R. (A) SDS–PAGE gel, stained with SYPRO Orange™, showing the interaction between either 100 μg pGST, GST 1–604, GST 1–225, GST 226–604 or 100 μg SCaBP1 in a pull-down assay in the presence of 200 μM free Ca²⁺. In lanes 2 and 3, SCaBP1 was retained by GST 1–604 and GST 1–225, respectively. (Bi) Map showing positions of synthetic peptides (A–F) used for binding experiments relative to the NH₂-terminal 159 aa region of the InsP₃R. Peptide A (S16-N48), peptide B (P49-N81), peptide C (Y66-K91), peptide D (D97-L123), peptide E (E106-S128) and peptide F (I121-L151). White bars indicating the Ca²⁺-independent CaM binding sites on the InsP₃R1 (Sienaert et al, 2002). Grey bar (B) showing a positive interaction of the peptide with SCaBP1. (Bii) Nondenaturating gel (15%) of 3.5 μM SCaBP1 in the presence of each of the InsP₃R1 peptides (A–F) (35 μM) in 50 mM Tris buffer (pH 7.4) containing either 200 μM free Ca²⁺ or 1 mM EGTA stained with SYPRO Orange™. In lane 1, SCaBP1 alone was used as a control. SCaBP1 bound to the peptide diminishes the intensity of the SCaBP1 band. (C) SDS–PAGE gel showing the interaction of SCaBP1 with increasing amounts of peptide B (P49-N81) in the presence of 200 μM Ca²⁺ or 1 mM EGTA stained with SYPRO Orange™. (Lower panel) Densitometric analysis of the SCaBP1 bands by ImageQuant 5.2 of the interaction between SCaBP1 and peptide B (P49-N81) in the presence of 200 μM Ca²⁺ (triangles) or 1 mM EGTA (squares). The vertical axis denotes the intensity of the SCaBP1 band. (D) co-IP of InsP₃R3 from COS-7 cells transfected with either SCaBP1 or LCaBP-YFP. YFP-CaBP1 was immunoprecipitated with anti-GFP antibody. InsP₃R3 immunoreactivity was detected using an anti-InsP₃R3 monoclonal antibody and subsequent ECL. Transfected cDNA is indicated above the blot. (E) co-IP of InsP₃R3 with SCaBP1-YFP was performed in the absence (lane 3) or presence of 1 mM EGTA (lane 4). Control IPs from YFP-transfected cells and nontransfected cells were also performed (lanes 1 and 2). (F) co-IP of CaBP with InsP₃R1 from the brain. The antibody used for the IP is indicated below the blot and the antibody used for the Western blot (WB) is indicated above the blot. C indicates IP with a non-specific antibody. (Fi) IP of InsP₃R1 subsequently detected by Western blot with anti-InsP₃R1 antibody. (Fii) Parallel IP using an anti-InsP₃R1 antibody and Western blot using an anti-CaBP antibody.