, and after MCAO were evaluated in the rat by a DRT-4 laser Doppler fluxmeter. The 20E-treated group was administered 20E at the onset of reperfusion, whereas the sham-operated group and operated group were given the same amount of distilled water. Preparation of cellular protein extracts The cells were harvested following treatment. After three washes with cold PBS, cells were lysed directly on culture plates with RIPA lysis buffer with protease inhibitors for 30 min on ice. The lysate was centrifuged at 140006g for 15 min at 4uC. The supernatant consisting of total cellular protein extracts was collected and stored at 70uC until use. The nuclear and cytoplasmic proteins were extracted according to the instructions of the nuclear and cytoplasmic protein extraction kit. In summary, cells were washed in ice-cold PBS and then were gently scraped off the bottom of the flask into the media using a cell scraper. The media were centrifuged for 5 min at 1200 rpm at 4uC, and the pellet was dissolved with cytoplasmic protein extraction agent A supplemented with PMSF. After vortexing for 5 s and incubation on ice for 15 min, the cytoplasmic protein extraction agent B was add into the tubes, which were then vortexed for 5 s and GW 501516 incubated on ice for 5 s. The samples were centrifuged for 5 min at 140006g at 4uC, and the supernatant, consisting of the cytosolic fraction, was immediately stored at 70uC until use. The pellet was resuspended in nuclear protein extraction agent supplemented with PMSF. After vortexing the tubes 1520 times for 30 min and centrifuging for 10 min at 140006g, the supernatants containing nuclear extracts were obtained. These protein extracts were quantified using a BCA Protein Assay kit. The nuclear and cytoplasmic proteins were subjected to immunoblotting to determine the activity of NF-kB in cells. Neurological deficits At 24 h after reperfusion, the neurological behavioral assessment was performed by an observer who was blinded to the experimental conditions in the surviving animals. Neurological deficits were scored on a 04 scale: 0 = no apparent deficits; 1 = contralateral forelimb flexion; 2 = decreased grip of contralateral forelimb while the tail was pulled; 3 = spontaneous movement in all directions, contralateral circling if pulled by the tail; 4 = spontaneous contralateral circling. Western blot analysis Equal amounts of protein were fractionated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and subsequently transferred to a nitrocellulose membrane. The membrane was incubated in fresh blocking buffer at room temperature for 1 h and then incubated with primary antibodies overnight at 4uC. The primary antibodies used in this study were anti-iNOS, anti-phosphorylated NF-kB , anti-phosphorylated IkBa, antiASK1, anti-phosphorylated ASK1, anti-MKK4, anti-phosphorylated MKK4, antiMKK7, anti-phosphorylated MKK7, antiJNK, and anti-phosphorylated JNK. The blots were washed three times in TBS-T for 5 min and then incubated with Measurement of infarct volume Animals were anesthetized with pelltobarbitalum natricum and sacrificed by decapitation 24 h after reperfusion. Brains were quickly removed, and five 2-mm consecutive coronal slices were made beginning from the anterior pole. The sections were stained with 2% TTC in saline 
for 30 min at 37uC and photographed. Then photographs of the five sections were analyzed for infarct 20-Hydroxyecdysone Inhibits Cerebral Injury size by a blinded observer using image analys
