Horseradish peroxidase conjugated anti rabbit secondary antibody

The T HESCs were lysed using lysis buffer (Jubiotech, Korea) containing protease and phosphatase inhibitor (Roche, Swiss). The protein concentrations were measured using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Proteins in cell lysate were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The blots were then blocked with 5% skim milk (Difco, USA) and probed overnight with primary antibodies at 4 ℃. The primary antibodies used in this study included FOXO1 (1:1000, cell signaling, #2880), NOX4 (1:1000, Abcam, ab109225), and GAPDH (1:5000, cell signaling, #5174). After probing with primary antibodies, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:5000, Millipore, AP132P). The expression of the target proteins was determined using an enhanced chemiluminescence kit (Thermo Fisher Scientific).

When the rats were sacrificed, the hippocampus was separated from the brian immediately and snap frozen in liquid nitrogen. Proteins were extracted from the hippocampus. Protein extracts were immunodetected with specific primary antibodies for the detection of BDNF (1:1000; Merck Millipore Corporation, Darmstadt, Germany) and for the detection of β-actin (1:5000, Sigma-Aldrich, Milwaukee, USA), which was used to evaluate protein loading in each lane. After a 12 h incubation at 4°C, the membranes were washed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (Millipore; 1:5000 with TBST) for 1 h. Finally the images were captured by densitometry (Bio-Rad, Hercules, USA).

Frozen NPC or non-tumor tissue samples were homogenized in radioimmunoprecipitation assay buffer (Qiagen, Shanghai, China). Following centrifugation at 15000 × g, 4°C for 20 min, 70 μg protein samples were run on a 12.5% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, St. Charles, MO, USA). Subsequent to blocking non-specific binding sites for 60 min with 5% fat milk, the membranes were incubated with rabbit monoclonal antibodies against CHD1L (1:1,000; Millipore) and GAPDH (1:1,000; Millipore) at 4°C overnight, respectively. The membranes were then washed with Tris buffered saline with Tween 20 (TBST) three times, for 15 min each time, and incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:10,000; Millipore) for 60 min at room temperature. The membrane was developed by an enhanced chemiluminescence system (Millipore) following washing with TBST three times. The intensity of the protein bands was determined by densitometry using Image J software (National Institutes of Health, Bethesda, MD, USA).