Figure 9

NMDA-induced caspase-3 activation is suppressed by 3-MA. A) Representative immunoblot of αII-spectrin breakdown profile shows the presence of the caspase-3 specific αII-spectrin breakdown product (SBDP) of 120 kDa in NMDA-treated cultures after 24 hours compared to the controls and NMDA+3-MA, or staursorpine (0.5 μM) (n = 3). Representative immunoblot probed with anti-SBDP120 shows a similar profile of the breakdown product in NMDA-treated cultures after 24 hours following treatment but not in controls or NMDA+3-MA co-treatment (n = 3). B) Densitometric analyses of the immunoblots probed with anti-SBDP120 show a significant increase in the αII-spectrin breakdown product of 120 kDa (SBDP120) after 24 hours following NMDA or STS treatment, when compared to control (* p < 0.05, paired Student T-test). NMDA+3-MA co-treatment significantly suppressed SBDP120, when compared to NMDA treatment alone (^#p < 0.05, paired Student T-test). The expressed values are means ± S.E.M. (n = 3) C) Caspase-3 enzymatic assay was determined using the caspase substrate Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) incubated with protease-inhibitor free lysates obtained from cultures treated with or without NMDA and NMDA+3-MA co-treatment at 24 hours. The expressed values are means ± S.E.M. (n = 3). Caspase-3 activity in NMDA or STS treatment are significantly higher than corresponding controls (* p < 0.05, paired Student T-test). NMDA+3-MA co-treatment significantly suppressed caspase-3 activity, when compared to corresponding NMDA treatments alone (^#p < 0.05, paired Student T-test).