Ab236870

Brain tissues were extracted, lysed and centrifuged at 12,000 r/min to remove tissue fragment or cell debris. After that, a bicinchoninic acid kit (Beyotime Biotechnology, Shanghai, China) was utilized for protein concentration determination. Next, 50 µg protein was collected and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane. Afterwards, the membrane was sealed with 5% skimmed milk and incubated with the diluted primary antibodies: Beclin I (ab62557, 1:500), LC3-I (ab48394, 1:1000), LC3-II (ab51520, 1:3000), NGF (ab6199, 1:1000), Nestin (ab6142, 1:1000), NeuN (ab236870, 1:1000) (all from Abcam, Shanghai, China), and GAPDH (1:500, ab8245, Abcam, Cambridge, UK). On the following day, the membrane was further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. After that, the protein bands were viewed with an enhanced chemiluminescence reagent (BB-3501, Amersham Company, Piscataway, NJ, USA), which were imaged on Bio-Rad image analysis system (Bio-Rad Laboratories, Hercules, CA, USA). Image analysis was followed using Quantity One v4.6.2 software. The target proteins were quantified as relative gray values against the internal reference GAPDH.