Western Blot Analysis of Protein Expression

Total protein from HTR-8 cells was prepared using 1× sodium dodecyl sulfate (SDS) lysis buffer (60 mmol/L Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, sodium salt, 0.01% bromophenol blue, 100 mmol/L DL-dithiothreitol). Then, the mixture was heated in a water bath for 8 min at 100°C. Equal amounts of protein samples were subjected to 12% polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Richmond, CA, USA). Then, 5% non-fat milk in Tris-buffered saline with Tween (TBST) was used to block the PVDF membranes for 1 h at room temperature. The membranes were incubated with antibodies against STMN1 (1:2000; Abcam), TTP (1:500; Abcam), p53 (1:1000; Cell Signaling Technology), p-p53 (1:1000; Cell Signaling Technology), p21 (1:1000; Cell Signaling Technology), α-tubulin (1:1000; Yeasen, Shanghai, China), or GAPDH (1:1000; Cell Signaling Technology) overnight at 4°C, followed by horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Yeasen) for 1 h. Signals were detected using a Prolight HRP chemiluminescent kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions [27 (link)].