Sds page loading buffer

For immunoblotting, whole cell extracts were lysed for 20 min at 4 °C using a RIPA lysis buffer (Beyotime, China) containing 1% protease and phosphatase inhibitor cocktail. They were then mixed with SDS‐PAGE loading buffer (Biosharp, China) and boiled at 100 °C for 5 min. Solubilized proteins were subjected to SDS‐PAGE and western blot analysis. For co‐IP, whole cell extracts were dispersed in 1 mL of cell lysis buffer (Beyotime, China) containing 1% protease inhibitor cocktail at 4 °C for 20 min, and then Anti‐Myc or Anti‐Flag or Anti‐HA immunomagnetic beads (Biomake, China) were added into solubilized proteins and waved at 4 °C overnight. On the second day, the immunomagnetic beads were collected by a magnet, washed three times, and then mixed with loading buffer and boiled at 100 °C for 5 min, finally followed by an immunoblotting procedure. Antibodies against DDRGK1, NRF2, KEAP1, and CUL3 were purchased from ProteinTech (China), the ß‐actin antibody was purchased from Affinity (USA), and PI3K, phosphorylated PI3K, AKT, phosphorylated AKT, cleaved caspase 3, and the PARP antibody were purchased from Cell Signaling Technology (USA).

Enzyme-linked immunosorbent assay (ELISA) kits for interleukin-1 (IL-1), IL-6, superoxide dismutase (SOD), etc. (Nanjing SenBeiJia Biological Technology Co., Ltd.), RIPA lysis buffer (Beyotime Institute of Biotechnology), SDS-PAGE loading buffer, protease inhibitor and bicinchoninic acid (BCA) protein assay kit (Biosharp), β-actin and secondary antibodies (Beijing Ray Antibody Biotech), primary antibodies (Cell Signaling Technology), tissue homogenizer (HaimenAiband Laboratory Equipment Co., Ltd.), EPS 300 electrophoresis apparatus (Bio-Rad, USA), Multiskan MK3 microplate reader (Thermo Fisher Scientific, USA), 2500 gel imager (Bio-Rad, USA), quantitative polymerase chain reaction (qPCR) instrument (7900 Fast, Applied Biosystems, USA), TRIzol reagent and DEPC-treated water (Medical Discovery Leader), UltraPure Agarose, SuperScript III reverse transcription (RT) kit and SYBR qPCR Mix (ABI), and pipette (Eppendorf).

The pre-treated NUGC-4 cells were scraped and lysed with radioimmunoprecipitation assay (RIPA) buffer containing 1:100 complete^TM Protease Inhibitor Cocktail (Roche, United States) in 1.5 ml EP tubes on ice for 20 min. Then, the lysed samples were centrifuged at 12,000 rpm for 20 min at 4°C. After centrifugation, the supernatants were transferred to clean EP tubes and mixed with 1:4 volume 5 × sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (BioSharp Inc., Nanjing). The samples were placed in boiling water for 5 min to ensure protein denaturation. Protein was quantified using bicinchoninic acid (BCA) reagent (Keygen). Subsequently, equal amounts of proteins (15 μg) from each sample were added to SDS-PAGE to separate the target protein, and then transferred onto polyvinylidene fluoride (PVDF) blotting membranes (GE Healthcare, Germany). The polyacrylamide gels were prepared in advance using a PAGE Gel Fast Preparation Kit (Epizyme Inc., Nanjing). Next, the membranes were blocked with 5–10% skim milk for 1 h, then cut into strips and incubated with the primary antibodies overnight and subsequently with the secondary antibodies for 1 h. Protein bands were visualized according to the instructions of the PageRuler pre-stained protein ladder (Thermo Fisher Scientific, United States).