Fig. 5.

Multiple phosphorylations of IP₃R1 during oocyte maturation. (A–C) Immunoblots (IB) of oocytes lysates at the different stages of maturation were probed with the MPM-2 (A), phospho-S421 (B) and phospho-T799 (C) antibodies (upper panel), and after stripping of the blot, re-probed with IP₃R1 antibody (lower panel). A representative result of 2–3 independent experiments with similar results is shown. Thirty oocytes for MPM-2 and 100 oocytes for phospho-S421 and phospho-T799 analysis were used per lane, both here and elsewhere in this study. (D) IP₃R1 PKA (upper panel) and IP₃R1 reactivity (lower panel) in oocytes at the different stages (GV, GVBD and MII). One of two independent experiments with similar results is shown. Fifty oocytes were used per lane. The intensity of the PKA reactive band from GV oocytes was arbitrarily given the value of 1 and values in the other lanes were expressed relative to this band. Data are presented as mean±SEM (n=2). Bars with different superscripts are significantly different (P < 0.05). (B) Oocytes were cultured in the absence or presence of H-89 (25 µM) for 2hr and in the presence of IBMX. Lysates from 50 GV oocytes were probed with the phospho-PKA substrate (upper panel), and after stripping of the blot, re-probed with IP₃R1 antibody (lower panel).