Gtx31722

The AQP3 and beclin-1 expression were measured by adding the primary antibodies (AQP3: ab125219, Abcam, Cambridge, UK; beclin-1: GTX31722, GeneTex, Irvine, CA, USA), followed by the secondary antibody of goat Alexa Fluor 568-conjugated anti-mouse IgG (H + L) (A11004, Invitrogen, Waltham, MA, USA). Beclin-1 expression reflects autophagy. Nuclei were stained with DAPI (Fluoromount-G; Sigma, St. Louis, MO, USA). Fluorescence images were acquired by Olympus BX53 microscope and analyzed using ZEN software (Zeiss, Oberkochen, Germany)^{31 (link)}.

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)}.