Radioimmunoprecipitation assay lysis buffer

Total proteins were extracted from SOSP-9607 cells using radioimmunoprecipitation assay lysis buffer (Bioswamp) and quantified using a bicinchoninic acid assay kit (Bioswamp). Each sample containing 20 μg of protein was separated and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). The membranes were blocked using 5% skim milk and incubated with primary antibodies against glucose transporter 1 (GLUT1, MAB37348, 1 : 1000), hexokinase 1 (HK1, MAB37234, 1 : 1000), lactate dehydrogenase A (LDHA, PAB30703, 1 : 1000), vimentin (PAB40646, 1 : 1000), E-cadherin (PAB43792, 1 : 1000), PI3K (PAB30084, 1 : 1000 dilution), p-PI3K (PAB43641-P, 1 : 1000), Akt (PAB30596, 1 : 1000 dilution), p-Akt (PAB43298-P, 1 : 1000), mTOR (PAB30674, 1 : 1000 dilution), p-mTOR (PAB36313-P, 1 : 1000), or GAPDH (PAB36269, 1 : 1000) for 1 h at room temperature, followed by incubation with goat anti-rabbit IgG (SAB43714, 1 : 20 000) secondary antibodies for 1 h at room temperature. All antibodies were supplied by Bioswamp. GAPDH served as an internal reference.

Total protein content was extracted from 3 × 105 cells in each group using radioimmunoprecipitation assay lysis buffer (Bioswamp) and quantified using a bicinchoninic acid kit (Bioswamp) following the manufacturer's protocol. 20 μg of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies against CDC2 (Bioswamp, PAB30052, 1 : 1,000), B-cell lymphoma-2 (Bcl-2, Bioswamp, PAB30042, 1 : 1,000), Bcl-2-associated X (Bax, Bioswamp, PAB30040, 1 : 1,000), nuclear factor erythroid-2-related factor 2 (Nrf2, Bioswamp, PAB30175, 1 : 1,000), TrxR1 (abcam, ab124954, 1 : 5,000), apoptosis signal regulating kinase 1 (ASK1, Bioswamp, PAB36297-P, 1 : 1,000), phosphorylated (p)-ASK1 (abcam, ab47304, 1 : 1,000), and GAPDH (Bioswamp, PAB36269, 1 : 1,000) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-labeled goat antirabbit IgG secondary antibody (Bioswamp, PAB160011, 1 : 20,000) for 1 h at room temperature and visualized using a Tanon-5200 apparatus (Tanon, Shanghai, China). The band gray values were read using the TANON GIS software (Tanon). GAPDH acted as the internal reference.

The expression of apoptosis-related proteins caspase 3, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated x (Bax), and cytochrome C (Cyt-c) was detected using Western blot assay. Total protein content was extracted using radioimmunoprecipitation assay lysis buffer (Bioswamp) supplemented with protease and phosphatase inhibitors. A bicinchoninic acid kit (Bioswamp) was used for protein quantification. Proteins (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% skim milk, the membranes were incubated with primary antibodies against caspase 3 (Abcam, ab13847, 1:1000), cleaved caspase 3 (Abcam, ab2302, 1:1000), Bcl-2 (Abcam, ab196495, 1:1000), Bax (Abcam, ab182733, 1:2000), Cyt-c (Abcam, ab133504, 1:5000), and GAPDH (CST, 2118, 1:1000) overnight at 4°C, followed by incubation with goat anti-rabbit immunoglobulin (Ig)G secondary antibody (Bioswamp, PAB150011, 1:10000) for 1 h at room temperature. Immunoreactivity was visualized by colorimetric reaction using an enhanced chemiluminescence substrate buffer (Millipore, MA, USA). The membranes were then detected using a Tanon-5200 apparatus (Tanon Science & Technology Co., Ltd., Shanghai, China) and the band gray values were read.