CaMKII activity is required for the oxygen/glucose deprivation (OGD)-induced activation of Tiam1 in hippocampal neurons. (A) CaMKII inhibition blocks the OGD-induced increase in Tiam1 activity. Cultures were treated with 10 μmol/L KN62 or vehicle as shown and active Tiam1 was analyzed using GST-RacG15A pulldowns as in Figure 3. Representative blots show the levels of active (RacG15A-bound) and total Tiam1 in hippocampal neurons under control and OGD conditions with or without KN62. Graph shows pooled data presented as mean±s.e.m. OGD-induced Tiam1 activation is abolished when CaMKII is blocked with KN62. *P<0.05 (T-test). Vehicle, n=5 independent cultures; KN62, n=7 independent cultures. (B) Endogenous expression of CaMKII (~54 kDa) is higher in hippocampal neurons compared with cortical neurons. Representative blots show total endogenous CaMKII in cell lysates prepared from hippocampal and cortical cultures. GAPDH (~37 kDa) was the loading control. Graph shows CaMKII level normalized to GAPDH, presented as mean±s.e.m. *P<0.05 (T-test). Hippocampal neurons, n=6 independent cultures; cortical neurons, n=6 independent cultures. GST, Glutathione-S-transferase.