Anti fam46c

Total protein was collected by using RIPA lysis buffer for 30 min at 4°C containing protease inhibitors, and the homogenates were centrifuged at 12,000×g for 20 min at 4°C. 15 μl of proteins were separated by 10-12% SDS-PAGE and transferred into nitrocellulose membrane (Millipore, Billerica, MA, USA). After blocking with 5% fat-free milk overnight at 4°C, the blots were incubated with anti-FAM46C (1:1000; Cat No. ab222808; Abcam), anti-PTEN (1:1000; Cat No. #9552; Cell Signaling Technology, Danvers, MA, USA), anti-p27 (1:1000; Cat No. #2552; Cell Signaling Technology), anti-cleaved Caspase-3 (1:500; Cat No. ab2302; Abcam), anti-p-AKT (1:1000; Cat No. #9271; Cell Signaling Technology), anti-AKT (1:1000; Cat No. #9272; Cell Signaling Technology), and anti-GAPDH (1:2000; Cat No. #5174; Cell Signaling Technology) antibody overnight at 4°C. The blots were then incubated with secondary antibodies conjugated with HRP (1:1000; Cat No. A0208, A0181 and A0216; Beyotime Biotechnology, Shanghai, China) for 1 h at 37°C. Protein expression was assessed by an enhanced chemiluminescence (ECL) kit (ECL New England Biolabs, Ltd, Whitby, ON, USA) following the manufacturer’s instructions.

We resolved cell proteins by Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis prior to electroblotting onto a Polyvinylidene Difluoride membrane. We incubated the membranes with the following primary antibodies for 1 hour at approximately 25 °C: anti-FAM46C (1:1000 dilution; Abcam Cambridge, UK), anti-MYC (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (1:1000 dilution; Thermo Fisher Scientific, Rockford, IL, USA). The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG at a 1:5000 dilution or anti-mouse IgG at a 1:5000 dilution; Cell Signaling Technology), and the bound proteins were visualized using a chemiluminescence-based detection kit (ImmunoStar Zeta or LD; Wako, Osaka, Japan).

The prostate cancer tissues samples (1.5 cm × 1.5 cm x 0.3 cm) were fixed in 10% formalin for 10 min, dehydrated in a gradient of ethanol for 2 h, and then transparent, paraffin and embedded. The slides (4-7 μm) were deparaffinized, rehydration and antigen-retrieved, after which slides were blocked by 3% H₂O₂ for 10 min and incubated with anti-FAM46C (Abcam, Cambridge, MA, USA) or anti-PTEN antibody (Abcam) at 25°C for 1 h and then stained with horseradish peroxidase (HRP)-labeled IgG (Shanghai Long Island Biotec. Co., Ltd, China) at 25°C for 20-30 min. Subsequently, the sections were stained with diaminobenzidine (DAB), counterstained with hematoxylin for 3 min and washed in water for 10 min. The tumor cells with positive staining more than 25% were defined as FAM46C or PTEN high expression group and that less than 25% were defined as FAM46C or PTEN low expression group.