Figure 4.

Prx2 is phosphorylated by cdk5 in focal ischemia and causes neuronal death. a) Quantification of infarct volume in striatum overexpressing GFP (n=5), Wt (n=7), as well as T89A (n=5) or T89E (n=4) forms of Prx2 following injection of endothelin-1. AAV-mediated expression of constructs in striatum was detected by Western blot analysis using anti-Flag antibody. b) Analysis of pPrx2 level in striatum at the time points following endothelin-1 injection by Western blot. The membrane is representative of n=3 experiments and graph presents densitometry values of pPrx2 relative to Prx2. c) Inhibition of cdk5 attenuates pPrx2. Rats were unilaterally injected with AAV expressing DNcdk5 and then endothelin-1 into both sides of striatum. Total proteins from both sides of the striatum were analyzed for pPrx2 by Western blot at 24 hours after ischemia. Expression of DNcdk5 has been shown using anti-Flag antibody. The membrane is representative of n=3 experiments and graph presents densitometry values of pPrx2 to Prx2. ipsi, virus injected side; cont, non-injected side. “NC” represents non-stroked control animals. d) P35 deficient mice are resistant to focal ischemia. P35−/− (n=5) or Wt (n=7) mice were injected with endothelin-1 in striatum and the infarct volume was measured. e) Increased pPrx2 signal is attenuated in p35 deficient mice in focal ischemia. Sections from mice brains (Wt n=5; p35−/− n=3) at striatum level were probed with anti-pT89Prx2 antibody and rabbit Alexa Fluor 488 antibody (green) as secondary antibody. Relative amount of luminescence was assessed by normalizing the average intensity of signal emitted (20 counts/brain) in the endothelin-injected side to that of the non-injected side. Data is presented as mean ± SEM (*Student’s t-Test, p<0.05).