FIGURE 4.

Individual IP₃Rs in a tetramer are ubiquitinated and degraded. A, αT3-1 cells were incubated with 100 nm GnRH for 7 min, and microsomes were prepared, lysed, and incubated with anti-IP₃R1 to immunoprecipitate IP₃R1 or with anti-ubiquitin to immunoprecipitate ubiquitinated proteins. Immunoprecipitated proteins were then electrophoresed by 4% SDS-PAGE, transferred to nitrocellulose, and probed with either anti-IP₃R1 or anti-ubiquitin. The bracket indicates ubiquitinated IP₃R1 at ∼270–400 kDa and the arrow indicates unmodified IP₃R1 at ∼260kDa. B, αT3-1 cells were incubated with 100 nm GnRH for 0,7, 20, or 60 min. The cell lysates were then electrophoresed by blue native PAGE and probed with anti-IP₃R1. The arrows indicate the migration positions of tetrameric IP₃R1 at ∼1.1 MDa and immunoreactivity that corresponds in size to trimeric and dimeric IP₃R1 complexes. Also shown is the percentage of total IP₃R1 immunoreactivity at each time point that migrated in the trimer/dimer region (mean, n = 2). C, 7% SDS-PAGE of the same samples, probed with anti-IP₃R1 (lanes 1–4) and NT1 (lanes 5–8), which recognize the C and N termini of IP₃R1, respectively.