Western Blot Analysis of Protein Samples

Preparation of the protein sample was carried out as described previously [45 (link)]. The samples were separated by 4–12% Tris-glycine precast gel (KOMA BIOTECH, Seoul, Republic of Korea) and then transferred onto a polyvinylidene Fluoride membrane (Millipore, Billerica, MA, USA). Blocking of the membrane was performed with 5% non-fat skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The membrane was incubated overnight at 4 °C with corresponding primary antibodies, including anti-ANO1 (ab64085; Abcam, Cambridge, UK), anti-β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-cleaved PARP (551,025; BD Biosciences, Franklin Lakes, NJ, USA) antibodies and then was washed three times with TBST followed by 1 h of incubation with HRP-conjugated anti-secondary IgG antibodies (Enzo Life Science, Farmingdale, NY, USA) at room temperature. Visualization was performed using the SuperSignal™ Western Blot Substrate (Thermo Fisher Scientific, Waltham, MA, USA).