Mini gel electrophoresis apparatus

After treatment, CATH.a cells were scraped from polystyrene culture dishes and immediately placed in ice-cold RIPA buffer supplemented with protease inhibitor cocktail (P8340, Sigma–Aldrich) and phosphatase inhibitor cocktail (P5726, Sigma–Aldrich). Cells were homogenized by sonication and total protein concentration was estimated by a Pierce BCA protein assay kit (Rockford, IL). A total of 20 μg (approximately 20 μL) of protein was boiled for 5 min in an equal volume of 4% SDS sample buffer and was loaded into a 7.5 or 12% SDS-PAGE gel, and ran at 100 V for approximately 1 h on a Bio-Rad mini gel electrophoresis apparatus (Hercules, CA). The protein was then transferred to a nitrocellulose membrane (Li-Cor, Lincoln, NE) at 50 V for 90 min. Membranes were then blocked in a 1:1 solution of Li-Cor blocking solution (Lincoln, NE) and PBS for 1 h. Membranes were then incubated overnight at 4°C in PBS with primary antibodies to BDNF (1:2000) and alpha-tubulin (1:5000), and incubated with Li-Cor secondary infrared-labeled antibodies (IRDye 680LT 926-68022 at 1:10,000 and IRDye 800CW 926-32214 at 1:5000) for 1 h at room temperature in PBS with 1% SDS. Bands were visualized using a Li-Cor Odyssey system and analyzed using Li-Cor Odyssey software.