Fig. 1.
CaMKII is not active after ascidian egg activation. (A) KN93 (150 μM) has no effect on Cyclin B Y170A destruction. Example traces of control eggs and eggs injected with KN93, both expressing cyclin B1 Y170A, were activated with ionomycin and the decrease in fluorescence as a result of cyclin destruction recorded. (A′) Analysis of destruction rates expressed as t1/2 values ±s.e.m. for all the eggs recorded in A show there is no significant difference in the destruction rates in control and KN93-injected eggs (n in parentheses). (B) CaMKII assays on ascidian eggs sampled at the times shown after activation with ionomycin. CaMKII activity (expressed as a percentage of the value at time 0) shows no increase up to and including 16 minutes post-activation; the rise at 20 minutes is not significant (P=0.7) (n=3, 15 eggs used per time point). Error bars represent s.e.m. (C) The CaMKII assay is sufficiently sensitive. Varying numbers of eggs expressing CA CaMKII were harvested along with the same number of control eggs. Each sample was assayed for CaMKII activity and the results show activity is detectable at more than three times that in controls in four eggs. (D) Exogenously expressed CA CaMKII activity is inhibited by KN93. Three eggs expressing CA CaMKII were injected with KN93 (final concentration 150 μM) and assayed for CaMKII activity along with three control eggs and three eggs expressing CA CaMKII (n=3). Error bars represent s.e.m.