Protein extraction buffer

RAW264.7 cells were lysed in the ice-cold protein extraction buffer (Solarbio) for total protein isolation. The concentrations of protein were detected using a bicinchoninic acid assay kit (Thermo Fisher). The protein was mixed with loading buffer with a final concentration of 2 mg/mL and denatured at 100°C for 10 min. Next, 10-μL protein samples in triplicate were loaded onto sodium dodecyl sulfate-polyacrylamide gels, electrophoresed, and transferred onto polyvinylidene difluoride membranes (Solarbio). The membranes were blocked with 5% blocking buffer for 1 h and then incubated with primary antibodies against ANO1 (ab72984, 1: 1000 dilution, Abcam, Cambridge, UK) overnight and horseradish peroxidase-conjugated IgG (secondary antibody; ab6721, 1: 10 000 dilution) for 2 h. β-Actin (ab8227, 1: 3000 dilution, Abcam) was the loading control. The protein bands were visualized with ECL western blotting substrate (Solarbio) and exposed to X-ray films in the dark. The relative protein level of ANO1 was assessed by QuantityOne (Bio-Rad, Hercules, CA, USA). The entire experiment was performed 3 times.