Protein Expression Analysis by Western Blotting

The proteins were then extracted based on the manufacturer's instructions. Equivalent amounts of protein per sample were electrophoretically resolved on 10% polyacrylamide gels and transferred onto 0.2 mm nitrocellulose membranes. Proteins were probed overnight with primary antibodies against AQP3 (1:1000; ab125219, Abcam), beclin-1 (1:1000; GTX 31,722, GeneTex), LC3B (1:1000; CST 3868, Cell Signaling Technology, Danvers, MA, USA), actin (1:5000, MAB1501, Millipore, Burlington, MA, USA). The nitrocellulose membranes were then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and the immunoreactivity was observed by enhanced chemiluminescence detection, a semiquantitative assay through blot dosimetry. Anti-β-actin (Epitomics, Cambridge, MA, USA) was used to check for equal loading of protein between wells^{25 (link)}.