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. Author manuscript; available in PMC: 2007 Sep 19.
Published in final edited form as: Curr Neurovasc Res. 2006 Feb;3(1):25–39. doi: 10.2174/156720206775541741

Fig. (7). Maintenance of an intact Rb/E2F1 complex during blockade of Rb phosphorylation is neuroprotective during NO exposure.

Fig. (7)

(A) A representative Western blot for Rb phosphorylation and E2F1 expression is illustrated for analysis at specific time periods following application of the NO donor NOC-9 (300 μM) using 50 μg/lane for neuronal lysates. The expression of p-Rb was significantly increased at 2 hr and continued over a 24 hr period following NO. E2F1 was significantly increased at 4 hr and continued over a 24 hr period following NO. (B) Primary hippocampal neurons were pretreated with the Rb phosphorylation inhibitors olomoucine (Olo, 50 μM) or butyrolactone (But, 50 μM) 24 hr prior to exposure to a NO generator (SNP, 300 μM or NOC-9, 300 μM)). Subsequently, the expression of p-Rb was determined at 4 and 6 hr following NO exposure. Representative Western analysis shown using 50 μg/lane for neuronal lysates. Olo and But significantly reduced p-Rb expression following NO exposure. (C) Representative results for E2F1/Rb immunoprecipitation are illustrated. Total protein extract was immunoprecitated by an antibody against Rb. E2F1/Rb complex expression by Western analysis reveals significant decreased expression of E2F1/Rb at 4 and 6 hr post NO exposure, but olomoucine (Olo, 50 μM) pretreatment significantly maintained E2F1/Rb expression at 4 and 6 hr following NO exposure. (D) Olomoucine (Olo, 50 μM) or butyrolactone (But, 50 μM) was applied to neuronal cultures 24 hr prior to exposure to a NO generator (SNP, 300 μM or NOC-9, 300 μM) and neuronal survival was determined by trypan blue exclusion 24 hr following NO exposure. Olo and But significantly increased neuronal survival from approximately 30% with NO only to approximately 70% (*p<0.01 vs. control untreated neurons; +p<0.01 vs. NO only treated neurons). (E) Olomoucine (Olo, 50 μM) or buty-rolactone (But, 50 μM) was applied to neuronal cultures 24 hr prior to exposure to a NO generator (SNP, 300 μM or NOC-9, 300 μM) and DNA fragmentation was determined by TUNEL assay 24 hr following NO exposure. Olo and But significantly decreased DNA fragmentation 24 hr following NO exposure (*p<0.01 vs. control untreated neurons; +p<0.01 vs. NO only treated neurons). In D and E, each data point represents the mean and SEM of n=4 determinations from four separate experiments. Control= untreated neurons not exposed to NO.