Chemiluminescent horseradish peroxidase substrate

Synchronized young adult worms were harvested and washed using M9 buffer and then centrifuged at 2000 g for 5‐10 seconds. More than 1000 worms (approximately 50 μL of worm pellets) for each condition were used for one set of sample. Worms were then immediately frozen at −80°C and mixed with 2× SDS sample buffer. The samples were boiled at 100°C for 10 min and were vortexed until the samples were broken. After 30‐min centrifugation at 15 000 g, supernatant was used for the assay. The worm lysates were electrophoresed using 8% SDS‐PAGE and transferred to PVDF membrane. The membrane was treated with 5% skim milk for blocking and subsequently incubated with primary antibodies against c‐Myc (Santa Cruz, Paso Robles, CA, USA; 1:1000) or α‐tubulin (Santa Cruz, 1:1000). The membrane was then incubated with goat anti‐mouse secondary antibody conjugated with horseradish peroxidase (Thermo, Waltham, MA, USA, 1:10 000). The PVDF membrane was then treated with the chemiluminescent horseradish peroxidase substrate (Thermo) for 1 min, and the signal was detected using X‐ray film (Agfa, Mortsel, Belgium). The band intensity was quantified using imagej (http://imagej.nih.gov/ij/).

Western blotting was performed as previously described [33 (link)]. BV2 cells were lysed on ice using radioimmunoprecipitation assay lysis buffer (ATTO Corporation), and the lysate was purified by centrifugation. For brain tissue, mice were killed, and 1 hemisphere was transferred to liquid nitrogen and stored at − 80 ℃. Protein extraction was performed by homogenization in cold PBS with 1% phosphatase inhibitor and 1% protease inhibitor cocktails (Thermo Fisher Scientific) using a homogenizer. Homogenates were extracted in lysis buffer and centrifuged for 20 min at 13,000 rpm. Protein concentration was measured using the Bradford assay. The samples were solubilized in 5 × sodium dodecyl sulfate buffer, boiled for 10 min at 100 °C, and loaded on 12–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels for protein gel electrophoresis. A pre-stained protein standard was used to determine molecular weights. Following electrophoresis, the samples were transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked in 5% non-fat skim milk powder in 1 × Tris-buffered saline with 0.05% Tween 20. The following proteins were analyzed by incubation with primary antibodies. The target proteins were then detected using a chemiluminescent horseradish peroxidase substrate (Thermo Fisher Scientific).