Bm3876

Tissues and cells are pyrolyzed in a protein lysis system containing RIPA, PMSF (Wuhan Boster Biological Technology, Ltd, Wuhan, China), and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein concentration was measured at 562 nm. A total of 30 μg of proteins were presented in SDS-PAGE gel system then separated and transferred to polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Bedford, MA, USA) for 90 min. After transfer to the PVDF membrane, the membrane was blocked in PBS containing 5% skim milk for 1 h and then incubated with antibodies against BMP1 (1:1000; ab205394; Abcam Co., Ltd) and GAPDH (1:2000; BM3876; Wuhan Boster Biological Technology, Ltd, Wuhan, China) at 4 °C overnight. The next day the membranes were washed and incubated with secondary antibodies (1:5000; BA1020; Wuhan Boster Biological Technology, Ltd, Wuhan, China) at room temperature (25 °C) for 2 h. Finally, the membranes were washed and detected by Biosense SC8108 Gel Documentation System with GeneScope V1.73 software (Shanghai BioTech, Shanghai, China).

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).