Protease inhibitor table

Cells were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) with a protease inhibitor table (Roche, Mannheim, Germany), and the protein concentration was detected by a BCA kit (Beyotime Biotechnology, Shanghai, China) as described elsewhere [26 (link)]. Targeted protein were migrated on a SDS-PAGE followed by transferring to PVDF membranes (Roche, Basel, Switzerland), followed by adding 5% nonfat milk. Then, the membranes were incubated with targeting antibodies overnight. After being incubated with appropriate horseradish peroxidase-conjugated secondary antibodies, the bands were detected by densitometry using the Tanon 5200 imaging system (Shanghai, China).

Protein extracts were prepared in RIPA buffer (50 mM Tris 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100) with protease inhibitor table (Roche). Blots were probed with anti–acetylated α-tubulin (1:1000, D20G3; Cell Signaling Technologies) and anti–α-tubulin (1:1000; Cell Signaling Technologies) and developed using either horseradish peroxidase–conjugated secondary antibody and WestPico Reagent (ThermoScientific) or IRDye infrared secondary antibody (LI-COR, Lincoln, NE). Quantitation was by scanning x-ray film and quantifying bands in ImageJ (National Institutes of Health, Bethesda, MD) or by integrated intensity using an Odyssey Infrared Imaging System (LI-COR).