Rabbit anti ano1

Cells were washed three times in ice-cold phosphate buffer solution and lysed in RIPA buffer with 1 X Halt phosphatase and protease inhibitor cocktails (Pierce, Rockford, IL). Samples were quantified using a BCA protein assay kit (Thermo Scientific, USA). Equal amounts of protein (50 μg) were separated using PAGE (8%) and transferred to nitrocellulose membranes. After blocked with Tris-buffered saline (TBS) containing 5% milk, the blotting membrane was incubated overnight at 4°C with rabbit anti-ANO1 (1:1000; Abcam) and rabbit anti -β-actin (1:500; Santa Cruz, CA). The membrane was incubated with an anti-rabbit lgG-HRP secondary antibody (1:5000, Santa Cruz) for an hour at room temperature and visualized using the Immobilon Western HRP Substrate.

The compound-treated NCI-H460 cells were cultured on 6-well plates were rinsed twice with PBS and harvested using a PRO-PREP™ lysis buffer (iNtRON, Seoungnam, Korea) that was supplemented with phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 8% gel and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in TBST for 1 h at room temperature. After that, the membranes were incubated with primary antibodies with 1% bovine serum albumin (BSA) in TBST at 4 °C overnight. The following primary antibodies were used: rabbit anti-ANO1 (1:1000, abcam) and rabbit anti-GAPDH (1:5000, sc-25778). Then, the membranes were washed 3 times for 10 min each and incubated with secondary antibodies (1:5000, GeneTex) for 1 h at room temperature. The membrane was then washed three times with TBST for 10 min and then visualized using the enhanced chemiluminescent (ECL) detection on an ImageQuantTM LAS-4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).