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).