Figure 7

Association of IP3R with the 150 kDa form of IRAG and effects of PKG or Src activity; IRAG not precipitated with IP3R1 binds cytoskeletal proteins. (a) Immune precipitation of IP3R1 followed by western analysis for IRAG. The ∼150 kDa form of IRAG binds IP3R1, consistent with other reports.⁷ In keeping with immune localization (Figure 3), the IRAG–IP3R1 complex was inhibited by NO donors. The Src antagonist PP2, but not its inactive congener PP3, also stabilized the complex, even when sodium nitroprusside (SNP) was added. A control western blot from total lysate confirmed similar quantities of large and small forms of IRAG in this preparation (lower panel). (b) Studies precipitating IP3R1, then blotting using antibody to IP3R1 (top panel), stripping it, and re-blotting for IRAG (third panel). Separate aliquots of the lysates were blotted directly for IP3R1 phosphotyrosine353 (second panel), followed by stripping and re-blotting for actin (bottom panel). Two cGMP inhibitors preserve IRAG–IP3R association (left two lanes) in preparations in which cell Ca²⁺ was held at a low level by BAPTA. In the NO donor sodium nitroprusside (SNP) and with the cGMP analog 8-Br-cGMP complexes were dissipated. With the NO donor or the cGMP analog, tyrosine phosphorylation of IP3R1 was observed (this is dependent on Src; see text and (c) below). (c) Immune precipitation of IP3R1 with western analysis for IP3R1, followed by stripping membranes and re-blotting for IRAG (top two panels). Separate aliquots of the supernatants were analyzed by western blot for phospho-Src tyrosine415; this blot was stripped and blotted for total Src and actin (bottom three panels). Strong Src phosphorylation occurred after sodium nitroprusside (100 μm), but the Src antagonist PP2 eliminated this and, as in (a), IRAG–IP3R1 association was retained in NO donor cells when Src activity was eliminated.