Phosphorylated protein kinase b p akt

Minimum essential medium (MEM) media and fetal bovine serum (FBS) were purchased from Gibco-BRL Co. (Grand Island, NY, USA). Penicillin, streptomycin, methylthiazolyldiphenyl-tetrazolium bromide (MTT), 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), vitamin C, H₂O₂, ethanol, acetylthiocholine, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), trichloroacetic acid, thiobarbituric acid, bovine serum albumin, dimethyl sulfoxide (DMSO), egtazic acid (EGTA), and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). An ENLITEN adenosine triphosphate (ATP) assay system was purchased from Promega Corp. (Madison, WI, USA). ProtinEX Animal cell/tissue, a tissue lysis buffer, was purchased from GeneAll Biotechnology (Seoul, Korea). Phosphorylated c-Jun N-terminal kinases (p-JNK), phosphorylated Tau (p-Tau), and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and phosphorylated protein kinase B (p-Akt) and Bcl-2-associated X protein (BAX) were purchased from Cell Signaling Technology (Danvers, MA, USA).

At 24 h after debridement, protein samples of the wounded corneas were harvested. For each group, six corneas form six mice were collected. Samples (20 μg) were run on SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 2 h and then incubated at 4°C overnight with the primary antibodies against hematopoietic prostaglandin D synthase (Hpgds) (Cayman, 160013), phosphorylated protein kinase B (p-AKT) (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 4691), phosphorylated signal transducer and activator of transcription-3 (p-STAT3) (Abcam, ab76315), STAT3 (Cell Signaling Technology, 4904), and β-Actin (Affinity, AF7018), followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

WB was performed using antibodies to VEGF (ab32152), nerve growth factor (NGF; ab6199), BDNF (ab108319), glial cell-derived neurotrophic factor (GDNF; ab18956), neural cell adhesion molecule 1 (NCAM1; ab9018), and growth associated protein 43 (GAP43; ab12274) purchased from Abcam, and to proliferating cell nuclear antigen (PCNA; #13110), protein kinase B (AKT; #4691), phosphorylated protein kinase B (p-AKT; #4060), CD31 (#77699), and GAPDH (#51332) purchased from Cell Signaling Technology.
Schwann cells and sciatic nerves were harvested from the experimental and control groups for WB. Briefly, Schwann cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Applygen) for protein extraction. Then, aliquots of 20 μg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), which were then incubated with antibodies as described above. Western blots were imaged using an Amersham Imager 600.