Phospho cdc2 thr161

Protein levels were analyzed in whole cell lysates that were obtained with RIPA lysis buffer (Beyotime, China), and 50 μg of each sample were resolved on a SDS polyacrylamide gel. Gels were analyzed by immunoblotting with antibodies against Cytochrome C, COX IV, Bcl-xL, Bcl-2, NDUFS1, NDUFS6, UQCRFS1, CYC1 (10993-1-AP, 11242-1-AP, 10783-1-AP, 12789-1-AP, 19532-1-AP, 12444-1-AP, 14417-1-AP, 18443-1-AP, 10242-1-AP, respectively; Proteintech; 1∶1000 dilutions), GAPDH, Bax, cleaved PARP, CyclinB1, Phospho-cdc2(Thr161) (2118S, 5023, 9541, 4138, 9114 respectively; Cell Signaling; 1∶1000 dilutions), H3P (ab5176, 1∶2000 dilutions) and acetylated histone H3 (06-599; Millipore; 1∶1000 dilutions). Immunoblotting analysis was performed with Enhanced Chemiluminescence (ECL) Western Blotting Detection reagents (Pierce).

At 6 hours after discontinuation of isoflurane, mouse pups were euthanized; the brains were immediately removed; and the cerebral cortices (an area particularly prone to neuronal apoptosis)6 were harvested, frozen in liquid nitrogen, and stored at −80°C. The samples were then lysed in RIPA buffer, and proteins were separated by SDS‐PAGE and electrophoretically transferred to a PVDF membrane. The immunoblots were probed with an anti‐cyclin B1 (Cell Signaling Technology, Danvers, MA, USA Catalog #4138). Phospho‐cdc2 (Thr161) (Cell Signaling Technology, Catalog # 9114), Phospho‐cdc2 (Tyr15) (Cell Signaling Technology, Catalog # 9111), phospho‐n‐myc (Bethyl Laboratories, Montgomery, TX, USA Catalog # A300‐206A), Bcl‐xL (Cell Signaling Technology, Catalog # 2762), Bax (Cell Signaling Technology, Catalog # 2772), Phospho‐Bcl‐xL (Ser62) (Thermo Fisher Scientific, Waltham, MA, USA Catalog # 44‐428G), Cytochrome c (Cell Signaling Technology, Catalog # 4272), and Cleaved Caspase‐3 (Asp175) (Cell Signaling Technology, Catalog # 9661). β‐Actin (Cell Signaling Technology, Catalog # 4967) was used as an endogenous control. Membranes were scanned and captured using an Odyssey detection system (LI‐COR Biosciences, Lincoln, NE, USA). Protein band intensities were quantified by densitometric analysis with ImageJ software and normalized according to endogenous control for each sample.