FIGURE 1.

SPFH1 and SPFH2 associate with activated IP₃R1 independently of polyubiquitination. A, αT3-1 cells were treated without or with 100 nm GnRH for 3 min, and cell lysates prepared in 1% Triton X-100-containing lysis buffer were incubated with anti-IP₃R1 to immunoprecipitate (IP) IP₃R1. IPs were subjected to SDS-PAGE and either Coomassie Blue stained (lanes 1–2) or probed in immunoblots (IB) for SPFH1 and/or SPFH2 as indicated (lanes 3–6). Arrows indicate the migration positions of IP₃R1, IgG heavy chain, SPFH1, and SPFH2. B, αT3-1 cells were treated with 100 nm GnRH for the times indicated, and anti-IP₃R1 IPs and control samples were probed for the indicated proteins. Quantitated data for IP₃R1 polyubiquitination and p97, SPFH2, and SPFH1 co-immunoprecipitation are graphed (n = 4). C, αT3-1 cells were incubated with or without 10 μm bortezomib (Bz) for 30 min, followed by treatment with 100 nm GnRH for 0–20 min, and anti-IP₃R1 IPs were probed for ubiquitin, SPFH1, and SPFH2. Quantitated data for IP₃R1 polyubiquitination and SPFH2 co-immunoprecipitation are graphed (n = 3). Data for SPFH1 were almost identical to that for SPFH2 (not shown).