Ab109446

After extracting protein using RIPA buffer (Beyotime, Shanghai, China), the protein was quantified using BCA Protein Assay Kit (Tiangen, Beijing, China). Then, the protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (Beyotime). Following blocking with 5% skimmed milk for 2 h at room temperature, the membranes interacted with primary antibodies against proliferating cell nuclear antigen (PCNA; 1:1000, ab18197, Abcam), Bcl-2 associated X protein (Bax; 1:1000, ab104156, Abcam), HDAC9 (1:20000, ab109446, Abcam), or GAPDH (1:2500, ab9485, Abcam) overnight at 4°C. After washing, the membranes were probed with HRP-coupled secondary antibody (1:25000, ab205718, Abcam) at room temperature for 2 h. Finally, the signal intensity was measured using ECL reagent (Absin, Shanghai, China).

Cells and tissues were treated with radioimmunoprecipitation assay lysis buffer (Abcam) to extract total protein and treated with bicinchoninic acid (Beyotime) to quantify protein concentration. After 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis-dependent separation, protein samples were transferred to polyvinylidene fluoride membranes (Bio-Rad, China) and blocked with 5% bovine serum albumin. After these steps, the membranes were treated with primary antibodies diluted with blocking buffer in a shaker at 4 ^∘C overnight. The next day, the primary antibodies were washed away with Tris-Buffered Saline Tween (TBST), followed by incubation of the membranes with specific secondary antibodies in a shaker for 2 h. After another wash with TBST, signaling was detected with the enhanced chemiluminescence kit, using NIH Image J software (version 1.52a; National Institutes of Health, Bethesda, MD, USA) to analyze gray-scale values. The antibodies used in this assay were as follows: HDAC9 (1:10000, ab109446, Abcam), VEGFA (1:450, ab183100, Abcam), GAPDH (1:2500, ab9485, Abcam), and secondary antibody (1:2000, ab205718, Abcam).