Tissue total protein extraction kit

Proteins from the tumor tissue were extracted with a tissue total protein extraction kit (SolarBio Tech Co., Ltd., Beijing, China) by homogenization at 4°C, and they were quantified by a bicinchoninic acid (BCA) protein assay kit (CoWin Biosciences Co., Ltd., Beijing, China); 30 μg proteins were separated by polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. Subsequently, the membrane was blocked with 5% skim milk in TBST for 1 h and incubated with the primary antibodies overnight at 4°C and with secondary antibodies for 1 h the next day. The protein bands were detected with the enhanced chemiluminescence (ECL) detection reagents (SolarBio Tech Co., Ltd., Beijing, China), and the band intensity was quantified using ImageJ software (NIH Image, United States).

We used a tissue total protein extraction kit (Solarbio, China) to extract the total protein in spinal tissues and detected the concentration of total protein using a BCA kit (Solarbio, China). The total proteins were analyzed by 10% SDS-PAGE followed by being transferred to the PVDF membrane. After blocking with 5% skimmed milk for 1 h at room temperature, the primary antibody was added and incubated overnight at 4°C. The PVDF membrane was washed three times with PBS buffer followed by incubation with secondary antibodies for 1 h at room temperature. The protein bands were visualized after adding ECL solution (Solarbio, China). The information about the antibodies used in the present study is shown in Table 1.