Sds page sample loading buffer

For the tissue-based assay, the brains of three mice in each group were homogenized in RIPA buffer (Beyotime, Jiangsu, China) by a hand-held motor and kept on ice for 30 min to lyse the cells completely. The homogenates were then centrifuged at 14,000 × g at 4°C for 15 min. The supernatants were collected and protein concentration was determined using a BCA protein assay kit (Boster, Wuhan, China). Equal protein was mixed with 5 × SDS-PAGE sample loading buffer (Beyotime, Jiangsu, China) and boiled for 5 min at 99°C.
For the cell-based assay, cells were lysed with RIPA buffer (Beyotime, Jiangsu, China) according to the manufacturer’s instructions. Protein concentration was determined by a BCA protein assay kit (Boster, Wuhan, China) and equal protein concentrations were mixed with 5 × SDS-PAGE sample loading buffer (Beyotime, Jiangsu, China) and boiled for 5 min at 99°C.
All tissue and cell samples were fractionated using 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). Membranes were incubated with primary antibodies overnight at 4°C, detected with HRP-conjugated secondary antibodies (Zhongshan Jinqiao Biology) and developed using an ECL chemiluminescence kit (Millipore). The optical density (OD) of each band was determined using Gel Pro Analyzer 6.0 (Media Cybernetics, Bethesda, MD, United States).

Western blot analysis was performed as previously reported [50 (link)]. Protein extracts of the tissues and cells were obtained using ice-cold RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor [Beyotime, Shanghai, China]) containing phenylmethanesulfonyl fluoride (PMSF, Beyotime). Protein extracts were boiled in 5× SDS-PAGE sample loading buffer (Beyotime) for 10 min, and protein concentration was determined using a BCA protein assay kit (Beyotime). Up to 15 µg of protein were separated in SDS-PAGE gels (Beyotime) by electrophoresis, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Solarbio, Beijing, China). After membrane blocking in 5% nonfat milk for 2–3 h, the membranes were incubated with the primary antibodies overnight at 4 °C and then washed three times for 30 min in TBST (Tris-buffered saline and Tween-20) before incubation with the secondary antibodies (ZB-2301; Beijing Zhongshan Golden Bridge Technology Co., Ltd., Beijing, China) conjugated with horseradish peroxidase for 2 h at room temperature. The membranes were washed three times with TBST and further quantified using chemiluminescence with a super enhancer ECL kit (Novland, Shanghai, China). The quantification of western blot bands was performed using ImageJ. All experiments were performed at least three time.