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Fig. 6. (A) Injection of phosducin or ßARKct prevents normal Ca2+ release at fertilization. Each data point represents an average of 6 or 8 recordings (error bars, 1±s.d.). (B) Delay of Ca2+ release during fertilization of sea urchin eggs injected with 80 µg/ml phosducin. Fluorescence intensity over the individual eggs (injected with the indicated reagents) is shown over time, with the number of recordings in parentheses. Asterisks over traces indicate gamete fusion. Phosducin-injected eggs exhibited either a dramatically delayed and attenuated Ca2+ transient (top trace) or a mildly delayed and attenuated Ca2+ transient (bottom trace). The average delays for each group are summarized in below; *, values are significantly different from the denatured control by non-parametric Wilcoxin Rank Sum test, P<0.02. (C) Injection of inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] overrides phosducin effects and causes cortical granule exocytosis (CGE). Two eggs were injected with 80 µg/ml phosducin with Rhodamin dextran (red fluorescence). After 1 hour incubation, the left egg was injected with 28 nM Ins(1,4,5)P3 with Alexa Fluor488 dextran (green fluorescence; orange when overlaid with red), which caused CGE and formation of fertilization envelope (FE). Arrowheads indicate oil droplets resulting from microinjection. (D) Response of phosducin-injected eggs to suboptimal amounts of Ins(1,4,5)P3 (10 nM final concentration) is indistinguishable from that of control eggs. Shown are the results of a representative experiment; 6-15 eggs were analyzed per group. One hour after microinjection of the control or test reagent, the eggs were challenged with 10 nM Ins(1,4,5)P3, and the time to initiation of CGE was monitored (white diamonds: the average time for the group; error bars, 1±s.d.). Black bars represent the percentage of eggs that were able to initiate CGE within 1 minute of Ins(1,4,5)P3 microinjection.
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