Tris glycine precast gel

Cell extracts and immunoblotting were prepared as described previously [23 (link)]. FRT-ANO1, PC3, CFPAC-1 and A549 cells were lysed with cell lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, and protease inhibitor mixture). Whole cell lysates were centrifuged at 15,000 g for 10 min at 4°C to remove the cell debris, and equal amounts (80 μg protein/lane) of supernatant protein were separated by 4–12% Tris-glycine precast gel (KOMA BIOTECH, Seoul, Korea) and then transferred onto PVDF membrane (Millipore, Billerica, MA). Membrane was blocked with 5% non-fat skim milk in Tris-buffered saline (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) including 0.1% Tween 20 for 1 hour at room temperature. This membrane was then incubated overnight with primary ANO1 antibody (a generous gift of Young Duk Yang, CHA University). After washing with 0.1% Tween 20 in Tris buffered saline (TBST), the blot was further incubated for 45 min at room temperature with an anti-rabbit secondary antibody (Cell Signaling). The membrane was then washed three times with TBST for 5 minutes and then visualized using the ECL Plus western blotting detection system (GE Healthcare Amersham; Piscataway, NJ).

A2058 and MEG-01 cells were plated and grown overnight on 6-well plates. For sample preparation, cells were lysed with RIPA lysis buffer (EMD Millipopre Corp., Billerica, MA, USA) supplemented with a protease inhibitor cocktail. Whole-cell lysates were centrifuged at 13,000× g for 20 min at 4 °C and the concentrations of supernatant protein samples were determined with a Bradford protein kit assay (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (70 μg protein/lane) were loaded and separated by 4–12% Tris-glycine precast gel (Koma Biotech, Seoul, Korea). The gel was then transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were washed with TBST (Tris-buffered saline with 0.1% tween 20) and incubated in blocking buffer (5% skim milk in TBST) at room temperature for 1 h. The membranes were incubated overnight at 4 °C with primary antibodies specific for PAR1 and β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1:1000). After three washes with TBST, the membranes were incubated for 1h at room temperature with an anti-mouse secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1:5000). The membranes were washed 3 times with TBST for 5 min each and then developed using the ECL Plus immunoblotting detection system (GE Healthcare, Piscataway, NJ, USA).

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).

Immunoblotting was performed as described previously [18 (link)]. FRT and PC-3 cells were lysed with cell lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, and protease inhibitor mixture). Whole cell lysates were centrifuged at 15,000 g for 10 min at 4°C to remove the cell debris, and supernatant protein was separated by 4–12% Tris-glycine precast gel (KOMA BIOTECH, Seoul, Korea) and then transferred onto PVDF membrane (Millipore, Billerica, MA). Membrane was blocked with 5% non-fat skim milk in Tris-buffered saline including 0.1% Tween 20 (TBST) for 1 hour at room temperature. The membrane was then incubated overnight with primary ANO1 antibody (1:500 dilution, ab64085; Abcam Inc., Cambridge, MA). After washing three times with TBST, the blot was further incubated for 60 min at room temperature with an anti-rabbit secondary antibody (Santa Cruz Biotechnology, CA). The membrane was then washed three times with TBST and then visualized using the ECL Plus western blotting detection system (GE Healthcare, NJ).

A549 cells stably expressing ΔF508-CFTR were lysed with cell lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, and protease inhibitor mixture). Whole cell lysates were centrifuged at 15,000 g for 10 min at 4°C to remove the cell debris, and equal amounts (80 μg protein/lane) of supernatant protein were separated by 4–12% Tris-glycine precast gel (KOMA BIOTECH, Seoul, Korea) and then transferred onto PVDF membrane (Millipore, Billerica, MA). Membrane was blocked with 5% non-fat skim milk in PBS including 0.05% Tween 20 for 1 hour at room temperature. This membrane was then incubated overnight with primary CFTR antibody (M3A7, Millipore, Billerica, MA). After washing with 0.05% Tween 20 in PBS (PBST), the blot was further incubated for 45 min at room temperature with an anti-mouse secondary antibody (Cell Signaling). The membrane was then washed three times with PBST for 5 minutes and then visualized using the ECL Plus western blotting detection system (GE Healthcare Amersham; Piscataway, NJ). The immunoblot results were analyzed quantitatively by ImageJ Software.

For Western blot analysis, HepG2 and Huh7 cells were lysed with RIPA buffer (50 mM Tris-HCl, PH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, and protease inhibitor). Lysed samples were centrifuged at 13,000 RPM for 20 min at 4 °C to remove the cell debris, and equal amounts (60 μg protein/lane) of supernatant protein were divided by 4–12% Tris Glycine Precast Gel (KOMA BIOTECH, Seoul, Korea). Then, PVDF membranes (Millipore, Billerica, MA, USA) were used to transfer the separated proteins. Membrane blocking was performed by Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% bovine serum albumin (BSA) at room temperature for 50 min. The membranes were incubated with primary antibodies overnight at 4 °C with the indicated primary antibodies: anti-cleaved PARP (BD Biosciences) and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). After incubating overnight, the membranes were washed out three times in 0.1% TBST and incubated with horseradish-peroxidase-conjugated secondary antibodies for 1 h. After 1 h, membranes were detected by using ECL Plus Western blotting detection system (GE Healthcare, Piscataway, NJ, USA).