Radioimmunoprecipitation assay buffer

MKN28 cells in the exponential growth phase were lysed by adding radioimmunoprecipitation assay buffer (Wuhan Boster Biological Technology, Ltd.). Following centrifugation at 30,000 × g at 4°C for 5 min, the supernatant was obtained to determine the protein level by the bicinchoninic acid method. Successively, 50 μg protein was extracted and mixed with 2× loading buffer prior (Wuhan Boster Biological Technology, Ltd.) to denaturation at 100°C for 5 min. Following separation by SDS-PAGE (Wuhan Boster Biological Technology, Ltd.), the proteins were transferred to a nitrocellulose filter (Wuhan Boster Biological Technology, Ltd.), where they bound to specific antibodies and relative secondary antibodies, were visualized by staining with enhanced chemiluminescence (Wuhan Boster Biological Technology, Ltd.) and were exposed, developed and fixed on X-ray films (Wuhan Boster Biological Technology, Ltd.). Gray-scale analysis was performed using BandScan software (ProZyme, Inc., Hayward, CA, USA).

Tissues and cells were pyrolysed in a protein lysis system containing radioimmunoprecipitation assay buffer (Wuhan Boster Biological Technology, Ltd.), a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) and PMSF (Wuhan Boster Biological Technology, Ltd.). Protein concentrations were measured with a bicinchoninic acid kit (Beyotime Institute of Biotechnology) at 562 nm. Thirty micrograms of each protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, Bedford, MA, USA) for 90 min. After the transfer, the membranes were blocked in phosphate-buffered saline with 5% non-fat milk for one hour and then incubated with antibodies against SLC4A4 (1:1000; A5332; ABclonal Biotech Co., Ltd.), KRAS (1:1000; A11059; ABclonal Biotech Co., Ltd.) and GAPDH (1:2000; BM3876; Wuhan Boster Biological Technology, Ltd.) at 4°C overnight. The next day, the membranes were washed and then incubated with secondary antibodies (1:5000; BA1020; Wuhan Boster Biological Technology, Ltd.) at room temperature (25°C) for two hours. Finally, the membranes were washed, and protein levels were detected with a ChemiDoc-XRS+ (Bio-Rad Laboratories, Inc., Hercules, CA, USA).