Ab169532

Total protein extraction was performed using a radioimmunoprecipitation assay solution. Protein concentration was measured using the BCA assay, and an equal quantity of each sample (100 μg) was used for Western blotting. Following SDS-PAGE, the proteins were transferred onto 0.45 µm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA). Membranes were blocked with 5% (w/v) non-fat dry milk in Tris base saline Tween 20 (TBST; 50 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween-20 (v/v)) for 1.5 hours, and then, washed thrice for 10 minutes in TBST, followed by incubation at 4 °C overnight with mouse anti-Alix (ab88743, Abcam), rabbit anti-CD9 (ab223052, Abcam), rabbit anti-Netrin1 (ab126729, Abcam), mouse anti-NF (#2836, Cell Signaling Technology), rabbit anti-microtubule-associated protein (MAP2) (#4542, Cell Signaling Technology), rabbit anti-β3-tubulin (#5568, Cell Signaling Technology), rabbit anti-Phox2b (25276-1-AP, Proteintech Group Inc, USA), rabbit anti-Hand2 (abs117611, Absin Bioscience Inc., China), GAPDH (10494-1-AP, Proteintech Group Inc), and Lamin A/C (ab169532, Abcam). Next, the membranes were washed thrice in TBST and incubated with a secondary antibody at room temperature for 2 hours. Following washing with TBST, the reaction was detected using chemiluminescence and the Western Bright ECL detection kit (WBKLS00100, USA).